BIOLOGY 204/205 Advanced Genetics Laboratory

TABLE OF CONTENTS

Introduction……………………………………………………………………………. p. 2MODULE 1: Recombinant DNA………………..…………………………………….

p. 13MODULE 2: Gene Expression………………………………..……………………….

p. 26MODULE 3: Screening a Plasmid Library Through Protein Expression………… p. 36MODULE 4: Detection of the ALU Insertion; DNA Fingerprinting……………… p. 40MODULE 5: Microarray……………………………………………………………….

p. 45Appendix A: Solutions Guide……………..………………………………………….

p. 68Appendix B: Sterile Technique………………………………………………………..

p. 81Appendix C: Spread Plate Technique………………………………………………...

p. 81Appendix D: Pipet Use……………………..………………………………………......

p. 82Appendix E: Pipet Exercises………………………….……………………………......

p. 84Appendix F: PCR Reagents and Conditions……………………………...………….

p. 85Appendix G: Pierce Protein Assay……………………….…………………………...

p. 86Appendix H: DNA/Protein Markers……………………………………………...…..

p. 87Appendix I: GST Plasmid Map……………………………...…………………….......

p. 88Appendix J: Streak Plate Method……………………………...……………….……..

p. 89Appendix K: SDS-PAGE Assembly Instructions…………………….…….………...

p. 90Appendix L: Western Blot Assembly Instructions…………………………………..

p. 91

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BIOLOGY 204/5Advanced Genetics Laboratory I and II

--- Introduction ---

Module 1 Recombinant DNA/Bacterial Transformation

This module gives you some of the experience you would receive if you were to sub-clone a gene as a part of your research. That is, once you transform a bacterial line with the plasmid that you isolate, you will need to demonstrate that you have made the transfer of the correct gene.

Goals:1. To purify a plasmid and transform E. coli with the plasmid.2. To demonstrate that the transformants carry the plasmid by

characterizing the transformants’ phenotypes. 3. Analyzing the size of the DNA plasmid in a cracking gel. 4. Hybridization with the original plasmid in a Southern blot.5. Amplify the gene inserted into the plasmid by PCR.6. Sequence part of the plasmid.

Module 2 Gene Expression

This module allows you to determine whether a cell is expressing a gene of interest, either GFP or GST. You will run the bacterial lysate on gels, stain with Coomassie blue to look for a protein of the correct size and perform a Western blot to determine whether the protein of interest was expressed.

Goals:1. Confirm, using PCR, that the plasmid from the transformed bacteria

(from module 1) has the GST gene.2. Show that bacteria with the gene for GST in the expression plasmid

are expressing GST using gel electrophoresis of the bacterial lysate.3. Show that GST is expressed by the bacteria using a Western blot of

the lysate.

Module 3 Screening a Plasmid Library Through Protein Expression

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This module lets you search for a gene of interest and demonstrate to us that you retrieved the right gene –not unlike the process that you would go through in the lab.

Goals:1. An antibody will be used to screen a human cDNA library for a clone

that expresses the particular gene product (in this case, GST).2. Positive clones will be isolated, plasmid will be isolated and restriction

digests will be done to determine the insert size.

Module 4 Human Genetics: Detection of the ALU Insertion; DNA Fingerprinting

This module allows you to carry out processes and procedures that a forensic scientist might carry out.

Goals:1. Isolation of your cheek cell DNA for polymerase chain reaction

amplification.2. Analysis of PCR products by agarose gel electrophoresis for the Alu

insertions in chromosome 8 and a polymorphism called a variable number tandem repeats (VNTR) D1S80 from the noncoding region of chromosome 1.

3. Use the techniques you learned to help solve a “crime” by comparing DNA “found” as evidence, and the DNA of the “victim” and the “suspects.”

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Biology 204/205 Advanced Genetics Laboratory Grading Policy

Biology 204 and Bio 205 are four credit courses. You will complete Modules 1 & 2 and a grant writing exercise during the fall semester for Bio 204. For Bio 205 in the spring semester, you will complete two modules (Modules 3, 4 if you have already completed 1 and 2) and Module 5.

The format of the course is a short introduction and two formal meeting times per week. The emphasis of the course is on experimental design, techniques, data gathering and analysis. Work at the bench is given priority over work in a lecture setting. The modules are designed to approach real situations in ongoing research projects. Therefore, the modules are not necessarily designed to be finished in three hours. A few labs will run long, taking 4-5 hours to finish. A few labs will be relatively short. Students will sometimes need to return on an alternate day, usually at their own convenience, to perform a short manipulation. Sometimes an experiment does not work and it has to be repeated. Coming to class well prepared and following directions carefully will cut down on potential mistakes!

Grading:Your grade will be based on the following components, each with approximately equal weight:

1. Performance in laboratory2. Discussion of experiments in class3. Notebook (these will be checked weekly) 4. Laboratory report 5. Grant proposal (Bio 204)6. Final oral exam covering both lecture and laboratory material

Supplies:You will need the following:

Laboratory manual (available through the Biology Department) Bound laboratory notebook UV safety glasses Sharpie permanent marker Transparent tape

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Note: It is important to read over the procedures in the laboratory manual and the corresponding information in the Molecular Biology Laboratory Atlas before coming to class. Be prepared to start work after an introduction by the instructor or TA. Check the laboratory calendar so that you know when each module will be done.

Laboratory Notebook Guidelines

Bound notebook; no loose-leaf Record in blue or black ink Number all pages Date all entries Name, course number and email address should be on front cover Reserve 3 pages at the beginning for the table of contents; keep up to

date Mistakes should be crossed out with a single line through the entry

then initialed Do not skip pages, do not rip pages out Unused portions of a page should have a diagonal line drawn through

the blank portion Each experiment should begin on a new page All data, calculations and graphs should be entered directly into the

notebook Neat, orderly, complete

Your notebook should provide enough detail so that another Advanced Genetics student could pick it up and repeat your procedure by following your entries. You should include all of the following information: What was done and why, who suggested it, who did it and when it was done, what results were obtained and what conclusions were drawn.

NOTE: NOTEBOOKS WILL BE CHECKED WEEKLY AND GRADED ACCORDINGLY!

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Laboratory Report Guidelines

You will be asked to write a formal report of one of the results from one of the lab modules. You will prepare this report as you would prepare a manuscript for publication, with introduction, methods, results, and discussion sections. To aid your preparation of this report, you should go to the library early in the semester and find a short article from Genetics from the last 5 years (download a pdf version or photocopy from a paper journal, the library has both formats). The format in Genetics is appropriate for your report. Below is a description of the content and length of each section.

The report in its entirety should not exceed 10 pages in length. It should be printed double-spaced, with no less than 1-inch margins. It must be in 12-point size in a common font. Each section except the introduction should be started by its section name, in bold type. At the head of the report, you should provide a title that indicates which exercise you are writing about and your name. Whenever possible, you should strive to write succinctly and in the active voice.

Abstract: 250 words summarizing the experiment.

Introduction: The introduction provides an overview of what the report is about, including why the exercise was done (the goal of the exercise) and an explicit statement of the hypothesis or hypotheses being tested. Background information about the biology underlying the exercise should be included in the introduction. Recommended length: 1.5 pages.

Methods: The methods section must be detailed enough to allow the reader to repeat the exercise. You do not need to repeat the detailed description of the protocols in the laboratory manual, but you should refer to the methods in the manual (Format: Laboratory manual Page x-y) at the appropriate points. Recommended length: 3 pages.

Results: The results section reports upon what happened during the exercise. You must include photocopies of the final gels and provide in tabular form other measurements and data you collected. Each figure should have a brief descriptive caption, and each table should have a title. However, it is not sufficient to simply insert these figures and tables. You must interpret your results in the text of the section, with references to the appropriate figure or table (Format: Fig. 1, Table 2A). Recommended length: 2 pages.

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Discussion: In the discussion, you should briefly re-introduce the main goal or hypothesis presented in the introduction, and then describe how your results are related to the goal or hypothesis. In subsequent paragraphs, you should discuss any failures to obtain results, and describe what you believe happened and what you would do differently to correct each problem. This is your opportunity to show how well you understand the molecular processes underlying the protocols! Recommended length: 2 pages.

Grant Writing Guidelines

The grant proposal must be based upon a novel concept that could be explored within the technological and financial limitations of our laboratory, using model organisms only. These limitations will aid you in narrowing your choice of topic.

A. Topic: The topic chosen should be novel, which means that no one else has worked on this same exact problem before. However it should also be one that can realistically attacked in the context of this laboratory. You should be able to put this topic into a broader context; Why is this an interesting problem? What has already been done with this problem in the past?

1. From this topic you must develop a testable hypothesis. This means you can develop an experiment that will result in data that leads you to clearly be able to reject or accept the hypothesis.

2. The experiment(s) will utilize techniques that you can do within this laboratory or with our collaborators.

3. If you are currently working in a laboratory outside of this class the topic you choose MUST be independent of that laboratory. You are not permitted to work on the same model organism or a topic that is related to your outside research.

4. You are permitted, indeed encouraged, to discuss possible topics with classmates, friends, family and other faculty.

B. Model Organisms: Your experiments must be limited to classic model organisms from molecular genetics that are readily available, easily maintained and for which we have appropriate technologies to care for and manipulate. Examples include: Drosophila, bacteria, Paramecium, C. elegans, yeast, small plants like grasses. You cannot work with organisms that are

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difficult and expensive to maintain (i.e. mice or other small rodents, fish, large vertebrates, humans, etc.)

C. Grant Format: Below is the format that your grant must follow. All text must be double spaced 12 point type with 1 inch margins. Make sure your grant contains all of the information within the guidelines given:

1. Cover Page - Fill out the cover page provided completely

2. Table of Contents - Page two is a table of contents. Provide the page number of each category. Number pages consecutively at the bottom right of each page throughout the application (including the cover page as page one).

3. Biographical sketch - A one page biographical sketch of the Principle Investigator (PI; this is you!). This contains your name, your date of birth, your education history, your previous relevant employment and a brief description of your prior experience that allows the reviewer to recognize you are capable of doing the proposed research.

4. Research Plan - This section should be 10 pages total. It should contain sufficient information needed to evaluate the project, independent of any other documentation. Be specific and informative, avoid redundancies. All tables, graphs, figures, diagrams and charts must be included within the 10 page limit. The following questions must be addressed in your research plan:

a. What topic will you be trying to address?

b. Why is this research important?

c. What has already been done in this field/topic?

d. What exact experiments will you be performing?

e. What are your expected results? (This is to clearly show your hypothesis. What results will allow you to accept your hypothesis? What results will cause you to reject your hypothesis?)

5. Literature Cited - All references cited in the proposal (of which there should be many) must be listed alphabetically by first author. They must include all authors, year of publication, complete title of article, journal name (no abbreviations or websites), volume and page numbers.

6. Appendix - In an appendix not to exceed 4 pages you may provide detailed descriptions of your protocols, with all appropriate references

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in your citations*. Your descriptions of the protocols should be as detailed as those provided in the laboratory manual.

*Protocols: If you are using protocols in the laboratory manual you may cite the manual itself as a reference. If, however, you propose other protocols you find and cite the original reference that describes the protocol.

Use the following format and page distribution for section 4 - Research Plan:

a. Specific Aims: Clearly state your hypothesis and then list the Specific Aims of your research to test the hypothesis. Specific Aims should organize the experiments that you will do to test the hypothesis. (See example below.) List the broad, long term objectives (goals) of the research and then present the proposed project's relationship to these goals. This section should not exceed one page.

b. Background Information: Summarize the major research that has been done on this topic leading to your proposal. Critically evaluate existing knowledge, and specifically identify the gaps. State where your research will fit in with what has been done previously, what new information does your research hope to discover? This section should not exceed two pages.

c. Research Design and Methods: Describe the overall research design and the specific procedures to be used. Include how data will be collected, analyzed and interpreted. Discuss the potential difficulties and limitations of the proposed procedures and alternative approaches to achieve the aims. Also provide a timeline for the project (not to exceed six weeks.) This section should not exceed six pages.

d. Possible Results: In terms of the hypothesis you are proposing to test, state what the results are expected. Which possible results will cause you to accept your hypothesis? Which possible results will cause you to reject your hypothesis? What are the limitations that may not allow you to have a clear answer? This section should not exceed one page.

This information was modified from the NIH instructions for US Department of Health and Human Services Public Health Grant

http://grants.nih.gov/grants/funding/phs398/phs398.html

Example: Hypothesis is that the moon is made of blue cheese.

Specific Aim 1: Build a rocket ship to get to the moon and tools to sample cheese.

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Specific Aim 2: Sample cheese on the moon and analyze it for its cheese-ness. Is it Blue or Velveeta?

Safety in the Laboratory

General Rules:1. Disinfect your bench top with a 10% bleach solution when you arrive

and when you finish lab.2. Wear gloves (lab coats, closed toe shoes and safety goggles are also

highly recommended). Avoid touching face and/or hair during an experiment. Chemicals on your gloves can be transferred to your face/hair.

3. Wash your hands before you leave lab.4. Do not eat, drink, smoke, chew gum or apply cosmetics while in lab.5. Dispose of all used materials as directed.6. Keep aisles clear.7. Wipe all spills immediately. Inform lab tech and/or TA if you spill

ANYTHING.

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8. Dispose broken glass in the appropriate receptacle. Inform lab tech and/or TA that you have broken glass.

9. Tie back long hair.10. Try to avoid wearing baggy, loose clothing that can interfere

with your experiment and may catch on fire. 11. Extinguish burners as soon as you finish using them.12. All Chemical Safety and MSDS information is located in the

binder on the back of the door.13. If you are unsure about a procedure, just ask.

General Instructions for Recombinant DNA Work

The recombinant DNA involved in Modules 1 and 2 requires a P1 level of containment. That means you must observe the following precautions:

1. Gloves must be worn to do the lab work.

2. Hands must be washed before leaving the laboratory

3. No eating, drinking or smoking in the laboratory.

Pipetting :

1. Acquaint yourself with the various denominations of pipettors in an attempt to avoid mistakes, particularly when working under time pressure.

2. The height of the fluid in the glass pipettes is measured at the bottom of the meniscus while the pipette is being held vertically.

3. Never put a pipette back into a sterile container.

4. Do not handle the lower part of the pipette.

Serial Dilutions: Serial dilutions allow you to dilute a sample many fold by making a series of small dilutions.

Standard Dilution Steps: Unless special circumstances demand it, the following are the only dilution steps that are used (For

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convenience and error avoidance in performing the accompanying arithmetic): 10, 20, 50 and 100.

10X 1:10 0.1 ml/0.9 ml 100 µL/900 µL

20X 1:20 0.1 ml/1.9 ml 50 µL/950 µL

50X 1:50 0.1 ml/4.9 ml 20 µL/980 µL

100X 1:100 0.1 ml/9.9 ml 10 µL/99 0µL

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Module 1 Recombinant DNA

Please refer to page 3 for introduction

**Note: The E.coli cell line used in this module is K12. The K12 bacteria cells are to be transformed with the GST plasmid.

1.0 Overnight (ON )Bacterial Culture (Done for you)

1. The lab tech will add 2.5 ml of cells previously grown ON to 125 ml LB amp medium (per group).

2. The cells will grow with shaking at 37C ON.

1.1 Isolation of Plasmid DNA HAZARDOUS CHEMICAL INFO: -Salt-Saturated Phenol is to be used only wearing gloves under the hood. Dispose of all pipets and liquid waste containing SS Phenol in appropriate disposals. -Chloroform: Isoamyl Alcohol (24:1) is to be used only wearing gloves under the hood. Dispose of all pipets and liquid waste containing C:IA in appropriate disposals.

1. Transfer 125ml of the overnight bacterial culture (Transformed E. coli) to a large, sterile centrifuge bottle and harvest the bacteria by centrifuging at 5000 rpm, 4ºC for 10 minutes in the Beckman J2-21.

2. Decant the supernatant broth into the waste jar.3. Resuspend the bacterial pellet in 5ml of Solution I containing 5mg/ml

lysozyme.4. Transfer to a 50ml polycarbonate Oakridge centrifuge tube. Let stand

at room temperature for 5 minutes.5. Add 10ml of freshly made Solution II. Place the cap on the tube and

mix the contents by inverting the tube several times. Let stand on ice for 10 minutes.

6. While tubes are on ice, put RNase A in 100°C heat block for 15 minutes. Allow to cool to room temperature before using in Step 11.

7. Add 8ml of ice-cold 5M potassium acetate (pH 4.8). Fill tubes only ¾ full. Screw on the cap and mix by inverting. Let stand on ice for 10 minutes.

8. Balance the tubes before centrifugation.9. Centrifuge in the Beckman J2-21; 15,000 rpm, 4ºC for 20 minutes.

The genomic DNA and bacterial debris should form a tight pellet at the bottom of the tube.

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10. Being very careful not to disturb the pellet, divide the supernatant in half. Transfer each half to separate 30ml glass tubes.

11. Add 500µL of heat treated RNase A to each tube.12. Incubate at 37ºC for 20 minutes.13. In the chemical flow hood, add one volume of SS (salt saturated)

phenol. (Note the yellow color which helps you identify the phenol phase in the next step.) Your tubes can be no more than 2/3 full including the addition of the phenol, so divide your original solution as necessary into 3 or 4 conical tubes. SAFETY NOTE: Phenol can cause severe burns to skin and damage clothing. Gloves, safety glasses, and a lab coat should be worn when working with phenol. All manipulations should be carried out in a fume hood. A glass receptacle is available exclusively for disposing of used phenol and chloroform.

14. Vortex the conical tube and contents with lids on for 1 minute; be sure the contents are thoroughly mixed. Make sure the tops of the conical tubes are screwed on tightly to ensure that no leaking will occur. Centrifuge for 1 minute at 2800 rpm using the IEC Centra 7 Benchtop.

15. Transfer the non-colored upper, aqueous phase to a fresh conical tube. Do not take the interface which is denatured protein. In the hood, add 1 volume of chloroform: isoamyl alcohol (24:1). Vortex 1 minute and centrifuge 1 minute at 2800 rpm.

16. Transfer the upper, aqueous layer to a fresh 30ml glass tube and add 2.5 volume of cold 95% ethanol. You need to calculate how much total liquid will be in each 30ml glass tube. The tube cannot be more than 2/3 full, so you may have to use more than one 30ml glass tube. Make your calculations before adding the ethanol!

17. Mix and allow to precipitate on dry ice for 15 minutes. 18. Balance your tubes along with their rubber sleeves.19. Recover the DNA by centrifuging the tube at 4ºC in the

Beckman J2-21 at 9500 rpm for 30 minutes.20. Discard the supernatant into a waste container. The pellet will

look like a whitish residue on the side of the tube. To resuspend the pellet, use the pipette tip to scrape the sides and then wash the residual ethanol up onto the sides of the tube using a Vortexer. Try to resuspend the entire pellet to increase your plasmid yield.

21. Start with one tube: wash the pellet with 1ml 70% ethanol by pipetting up and down to resuspend the pellet. Transfer the solution from the first tube to the resuspended pellet in another Oakridge tube and mix well. Transfer to the next tube until all pellets are resuspended and pooled together. Transfer the solution into one sterile 1.5ml microfuge tube.

22. Microcentrifuge for 5 minutes at 14,000 rpm. Discard the ethanol; add 1ml more of ethanol to wash the pellet. Spin at 14,000 rpm for 5 minutes.

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23. Discard the ethanol; dry the pellet using the SpeedVac in the basement. Give your sample to the TA/Lab tech to be properly dried.

24. Dissolve the pellet in 0.3ml TE. Aliquot 100 L to each of 3 microcentrifuge tubes (properly labeled!).

25. Store at -20ºC.

1. 2 Agarose gel to confirm isolation of the plasmid HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.-UV light is very harmful if looked at directly. When viewing your gels on the UV light box be sure to wear a protective face mask, or place the shield on top of the box before turning on light.

1. Prepare 250ml 1X TAE from 5X TAE stock.2. Dissolve 0.35g agarose in 50ml 1X TAE buffer to make a 0.7% gel.

3. Microwave on high for 1 minute.4. When done, swirl the flask and make sure all of the agarose is

dissolved. If not, microwave until it is.5. Place the running tray into the gel-casting tray. Add comb. 6. Cool agarose slightly, approximately 5 minutes; slowly pour into gel

casting set up (from the farthest corner away from the comb) with comb and let cool until opaque. Try to avoid bubbles!

7. While your gel is setting, thaw out one tube of your plasmid DNA on ice. Just before you are ready to load the gel, heat the λ Hind III marker for 7 minutes in the 65ºC hot block.

8. Mix 4µL of 6X DNA sample buffer with 20µL plasmid DNA on a piece of Parafilm.

9. Once your gel is set, remove it from the casting tray. Place it in the running tray, with the comb still set. Cover the gel with 1X TAE. Gently remove the comb. Removing the comb last will ensure that your wells do not collapse.

10. Be prepared to load the get quickly—you do not want your DNA to diffuse into the running buffer.

11. Load 24µL of plasmid DNA sample and 20µL of λ Hind III marker in the wells; put the lid on the box so that the DNA will run toward the red electrode.

12. Run the gel at 100V for ~1 hour.13. Stain the gel for approximately 7 minutes in ethidium bromide,

and destain in water for 4 to 5 minutes.

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14. Examine the gel on the UV light box. If the ladder is not visible or is faint, place the gel back into the stain. When you feel that your gel is properly stained, take a picture to document your results. (Make a photo copy for your lab partner.)

15. Leave the gel in destain or discard (your TA will inform you).

1.3 Grow an overnight broth culture of E. coli (Done for you)

1.4 Transformation

Three hours before class the tech will take 1 ml of an ON culture and inoculate 50 ml of fresh LB broth with it. It will shake at 37C for three hours. This will produce exponentially growing cells for you to transform.

1. Divide broth culture into 2 sterile 50ml Oakridge centrifuge tubes; place tubes in ice for 30 minutes.

2. Thaw out one tube of your plasmid DNA on ice.3. Centrifuge the cultures at 4ºC in the Beckman J2-21 for 7 minutes at

5000 rpm; decant the supernatant into the collection flask provided.4. Resuspend one pellet in 25 ml ice cold 50 mM CaCl2. Combine this

resuspension solution with the second bacterial pellet; place on ice for 20 minutes. Keep CaCl2 on ice while waiting.

5. Centrifuge the cell suspension at 4ºC in the Beckman J2-21 for 7 minutes at 5000 rpm.

6. Decant the supernatant and resuspend the pellet in 3ml ice-cold 50 mM CaCl2; place on ice for 5 minutes.

7. Dispense 2 aliquots of 0.3 ml cells in ice-cold microfuge tubes; add 0.2 ml of transformation buffer to each tube.

8. Add 5 µL [>2 g] plasmid DNA to one tube. The second tube will not contain plasmid DNA and will act as a control. Mix gently and leave on ice for 20 minutes.

9. Heat shock cells for 1 min in 42°C water bath.10. Let stand at room temperature for 5 minutes.11. Add 0.7 ml LB to each tube and tap gently with finger.12. Shake at 37ºC for 60 minutes.

**NOTE: during this hour incubation your TA or Lab Tech will demonstrate proper spreading and streaking procedures for plating. It is very important that you understand sterile technique when working with bacteria so you don’t contaminate your samples.

13. Plate 0.05, 0.1, and 0.3 ml of the cells with plasmid DNA onto LB amp plates. Use the spread plate technique.

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14. Streak (Do not use the spread plate technique) the contents of the “no DNA tube” on an LB amp plate and an LB plate. The LB amp plate will act as a negative control, while the LB plate will serve as a positive control.

15. Label plates appropriately with group number, date, type of bacteria, and any other important information, such as how much bacteria was plated.

16. Incubate the plates at 37ºC overnight (upside down); be sure to remove, wrap in Parafilm and refrigerate the plates tomorrow!

Following Day: 1.5 Selecting for bacteria that carry the plasmid________

1. Examine transformed and no DNA control plates. (There should be no colonies on the “No DNA” plate)

2. Choose 6 well isolated colonies from the transformed plates. Streak each colony on half of an LB amp plate.

3. Choose 2 well isolated colonies from the control (non-transformed) plate provided. Streak each colony on one half of an LB plate.

4. Incubate the plates overnight at 37ºC.5. Wrap the old plates in Parafilm and refrigerate.

1.6_Secondary selection of transformed bacteria_____________________

1. Transfer 4 well-isolated colonies from 4 different transformed streaks and 2 control colonies into separate 1ml aliquots of sterile saline. Refrigerate the old plates.

2. For the transformed bacteria, streak 1 loopful of saline/bacteria suspension onto ½ of an LB amp plate. Do this for each of the 4 samples.

3. For the control cells, streak 1 loopful of the saline/bacteria suspension onto ½ of an LB plate. Be sure to label plates clearly!

4. Incubate at 37°C overnight; remove and refrigerate the next day.5. Go to 1.16

1.7 Preparing bacteria for the cracking gel (day before 1.8)

1. Using a marker, draw a line down the center of a new LB amp plate. Make a template on paper with 1.5 cm x 1.5 cm squares on each half. Place the plate over the template.

2. Using sterile tweezers, select a sterile toothpick.3. Choose 2 LB amp plates from Day 1.6 that show the best growth.

With the toothpick, select one colony from the Day 1.6 plate and “fill in” the square on the agar on the plate. Repeat for the 2nd colony using a new toothpick.

4. Repeat the procedure for the control, but use a fresh LB plate.

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5. Incubate at 37ºC overnight for at least 24 hrs, but less than 36 hrs.

1. 8 Next day: Cracking gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 250ml 1X TAE.2. Prepare 0.7% agarose gel.3. Use a sterile toothpick to scrape bacteria from the plates prepared the

day before. Add bacteria from each square to 250 µL of cracking buffer (Two squares for one tube of 250 µL of cracking buffer). Do this for transformed and non-transformed cells (you should have a total of 2 microcentrifuge tubes).

4. Incubate at 37ºC in the hot water bath for 25 minutes.5. Centrifuge for 15 minutes at 14,000 rpm.6. Use a toothpick to remove the bacterial debris from the bottom of

each tube. (You won’t be able to see a pellet, but when you pull it out, it will look like a blue glob).

7. Load the gel: 8. Lane 1: 10 µL Hind III marker (Heat in 65C hot block for 7 minutes

before loading) Lane 2: 10 µL plasmid DNA solution (5µL plasmid DNA + 2µL 6X DNA sample buffer + 3µL 1X TAE)Lane 3: Transformed supernatant Lane 4: Non-transformed supernatant Lane 5: 50µL Cracking buffer only Note: Load as much transformed and nontransformed supernatant as possible (A well formed well can hold ~50 L).

9. Run the gel for 1 hour at 100 volts.10. Stain with ethidium bromide, destain, and photograph. Look for

genomic DNA, plasmid DNA and RNA.

1.9 Labeling DNA with Biotin Part A: Labeling Reaction

1. Remove an aliquot of Plasmid DNA from the refrigerator and place on ice.

2. Add labeling reaction components to a 0.5ml tube (on ice) in the following order:1X DNase I Buffer 19.9µLDNase I Enzyme 0.1µLdNTP mix 28µL

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Plasmid DNA 4µLdH2O 128µLDNA Polymerase I 20µL

3. Mix well and centrifuge for 5 seconds at 14,000 rpm.4. Allocate 50µL into 4 tubes.5. Incubate at 15°C for 2 hours in thermocycler.6. Add 5µL Stop Buffer to each tube and mix.7. Incubate tubes at 65°C for 5 minutes in thermocycler.

Part B: Purification of DNA probes1. Transfer liquid to consolidate solution from 4 tubes into one tube.2. Add 4µL 10% SDS to tube and mix.3. Add 110µL Chloroform and 110µL SS Phenol to an empty 1.5ml

microcentrifuge tube.4. Transfer DNA solution to chloroform phenol tube. Vortex 2

minutes and then centrifuge for 2 minutes at 14,000 rpm5. Collect the top layer of liquid and transfer to a fresh 1.5ml tube.

Discard remaining liquid into waste container.6. Add 220µL chloroform to tube. Vortex 2 minutes and then

centrifuge for 2 minutes at 14,000 rpm.7. Repeat step 5. 8. Add 40µL 3M Sodium Acetate (pH 4.8) and 800µL cold 95%

ethanol. Mix gently by inverting tube.9. Store at -20°C ON (at least 6 hours)

The Next Day:10. Centrifuge for 5minutes at 14,000 rpm.11. Carefully remove the supernatant.12. Resuspend the pellet in 1ml cold 70% ethanol. Centrifuge for 5

minutes at 14,000 rpm. 13. Remove supernatant (ethanol). Let tube dry in cabinet for at

least 1 hour. 14. Once dry, resuspend probe in 12µL TE buffer and store at -20°

C.

1.10 Preparing for the Southern Blot (day before 1.11)

1. Using a marker, draw a line down the center of the underside of a fresh LB amp plate. Draw two 1.5 cm x 1.5 cm squares on the underside of the plate, one on each half.

2. Using a sterile toothpick, pick one isolated colony from the Day 1.6 LB amp transformed plate. “Fill in” one square on the fresh LB amp plate with one colony. Repeat for the second square making sure to use a fresh toothpick.

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3. Repeat steps one and two, this time using an LB plate and the Day 1.6 non-transformed cells.

4. Incubate both plates for at least 24 hours.

1. 11 Southern Blot HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Run cracking gel (same as Day 1.8). Do not forget control lane! 2. Stain with ethidium bromide, briefly destain, and examine the gel. 3. Make sure to destain the gel for approximately 5 minutes before

denaturing.4. DO NOT cut the gel before running the southern blot. Nick a

corner of the gel before destaining for orientation purposes. Photograph the gel before destaining completely—you will use this photograph later to compare to the results of your southern blot.

5. Denature gel in 0.5 M NaOH/0.8 M NaCl for 30 minutes, rocking. Decant the solution and repeat.

6. Rinse gel in dH2O for 1 minute.7. While the gel is rinsing, cut and hydrate the nitrocellulose filter for 3

minutes in dH2O, then in 10X SSC until blot set-up is ready. Make sure to notch the corner of the nitrocellulose for orientation purposes and always wear gloves when handling the nitrocellulose. Always handle the filter with forceps, and only around the edges so as to not create blotches of background color.

8. Neutralize gel in 0.5 M Tris/1.5 M NaCl (pH 7.0) for 30 minutes, rocking. Decant the solution and repeat.

9. Rinse the gel in 10X SSC for 3 minutes, rocking.10. While the gel is neutralizing, prepare the Test Blot for 1.13.

a. Take your Biotin labeled probe out of the freezer and let thaw on ice.

b. While thawing, cut a small piece of nitrocellulose and hydrate in dH20 for 3 minutes.

c. Soak nitrocellulose in 10X SSC until the probe is thawed. d. Using 2 µL of probe, make a small dot on the filter.e. Dry in 80°C oven, then wrap in plastic wrap and store in

the freezer until 1.13.11. Assembling the Southern Blot:

-First the wick (a long strip of paper towel will work) needs to be placed on the platform so that it can only touch the buffer on two sides.

-Place three squares of Whatman 3M filter paper on top of wick.

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-The gel should be placed on top of the filter paper, and the nitrocellulose on top of that. **Make sure the nitrocellulose and the gel are lined up in the correct orientation so you can compare them later**

-Place three more squares of Whatman 3M filter paper on top of the nitrocellulose.

-A stack of cut paper towels at least 10 cm high should be assembled and tied together with string. This can go on top of the filter paper.

-Pressure should be applied to the top of the stack to enhance wicking overnight. (Your TA should demonstrate this and assist in the assembly)

12. Let Southern Blot transfer ON in 10X SSC.

1.12 Drying of Blot (Done for you)_________________________________

1. Disassemble blot and rinse nitrocellulose filter in 5X SSC for 2 minutes.

2. Dry on large Kimwipe.3. Bake filter in vacuum oven at 80ºC for 2 hours.4. Wrap in plastic Wrap and store in freezer.

1.13 Hybridization of the Southern Blot

1. Denature 200L of Herring sperm DNA (2mg/mL) by boiling for 10 minutes followed by fast chilling on ice.

2. While the Herring sperm DNA is boiling, soak the nitrocellulose filter (from Days 1.11/1.12) and the test spot (from Day 1.11) in 2X SSC until uniformly hydrated.

3. For prehybridization of the nitrocellulose filter, add the 200µL of freshly denatured Herring sperm to the prehybridization solution. Mix, and then transfer to two plastic tubs. Add the nitrocellulose filter to one, and the test spots to another tub.

4. Incubate at 42ºC for 2 hours. The volume of prehybridization solution used should be 20 to 100L per cm2 of the filter, ~7.5ml.

5. For hybridization, heat-denature the probe made on 1.11 and 200 L of Herring sperm DNA by boiling for 10 minutes in a boiling water bath and then place on ice. Just before use, add to the hybridization solution.

6. Remove the prehybridization solution from the tub and add the hybridization solution to the filter (20-100 L per cm2), ~7.5 ml. The filter should be hybridized at 42ºC overnight to achieve maximal sensitivity.

21

Following Day: 1.14 Detection of the DNA

**All the washes in this section need to be completed while rocking.**Decant and save the hybridization solution in an appropriate size tube. Store at 4C.

1. Wash the filter & test spots with 100ml of 2X SSC/0.1% (w/v) SDS at room temperature for 3 minutes. Decant the SSC and repeat.

2. Wash the filter & test spots with 100ml of 0.2X SSC/0.1 % (w/v) SDS at room temperature for 3 minutes. Decant the SSC and repeat.

3. Wash the filter & test spots in 100ml of 0.16X SSC/0.1% (w/v) SDS at 50ºC for 15 minutes. Decant the SSC and repeat.

4. Rinse the filter & test spots in 100ml of 2X SSC at room temperature for 1 minute.

5. Dry on large Kimwipe and then wrap in plastic wrap and store in refrigerator.

6. The hybridization mixture containing the biotin-labeled probe may be reused. Store the mixture at 4C for several days or at -20C for longer periods. Placing the hybridization solution in a boiling water bath and cooling on ice just prior to use should denature the probe.

1. 15 Development of Blot HAZARDOUS CHEMICAL INFO:-NBT/BCIP is highly toxic. WEAR GLOVES when handling and dispose of all liquid waste containing NBT/BCIP in the appropriate waste container.

1. Wash the filter and small test squares (from Day 1.11) in Buffer 1 at room temperature for 1 minute with sufficient buffer to cover the blots. Decant Buffer 1 into the sink.

2. Incubate filters in Buffer 2 in a plastic container (1 hour, 65ºC) with sufficient buffer to cover the blots.

3. Wash the filters in freshly made strep-avidin alkaline phosphatase (SA-AP) conjugate for 15 minutes at room temperature. (Add only enough SA-AP conjugate to cover the filters (~10ml). Use gentle agitation and occasionally pipette SA-AP over the filters.)

4. Decant and save the SA-AP in a 15ml tube. Save for step #8.Wash the filters in Buffer 1 using 20 to 40-fold greater volume than employed in step 3. Gently agitate filter for 15 minutes in Buffer 1.(ie., if you used 7 ml diluted SA-AP conjugate in step 3, wash with at least 140-280ml Buffer 1.) Decant Buffer 1 into the sink.

5. Wash the filters for 10 minutes in Buffer 3. Decant Buffer 3 into sink. 6. Add 1ml NBT/BCIP solution to the saved SA-AP. A blue color should

develop overtime. Wear gloves when working with NBT-BCIP.7. Decant Buffer 3 into the sink and add 9 ml of NBT-BCIP solution to

the filters. Allow the filters to develop for 15 minutes to 1 hour.

22

8. DNA bands will be most evident on only one side of the filter (check your notch for correct orientation). Check your filter every 2 minutes to ensure that over-development does not occur.

9. Once bands have developed, decant the NBT-BCIP solution in the appropriate waste container and wash the filter in TE. This will terminate the color development reaction. The TE can then be decanted into the sink.

10. To dry, bake at 80C in a vacuum oven for 1-2 hours. 11. Measure the photograph of the cracking gel, and compare the

relative position of the plasmid band to the results of the blot. Interpret your results.

12. Wrap filters in plastic wrap and label. The lab tech will photograph and distribute the gels for your notebooks.

1.16 Designing Primers 5’-------------------------------------------------------------------------------------------------------3’ 698 bp

GST

Partial DNA Sequence for GST (Read left to right, top to bottom):

5’……GTATTCATGTCCCCTATACTAGGTTATTGAAAATTAAGGGCCTTGT

310GCAACCCACTCGACTTCTTTTGA……….…ATCCTCCAAAATCGGATCTGGT

960TCCGCGTGGATCCCCGGGAATTCATCGTGACTGACTA………….……………..3’

The glutathione S-transferase protein consists of 232 amino acids. The sequence—using the one-letter abbreviation for each amino acid—is shown below.

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPGIHRD

Using this information, design the primers to amplify the GST gene. Once you have designed the primers, fill out the oligonucleotide request form. The primers will then be made on a DNA synthesizer.

23

1. 17 PCR

1. Set up 7 - 0.5 ml PCR reaction tubes according to the PCR chart in Appendix F. Read the chart carefully and make sure you add the correct amounts of reagents. PCR is a very sensitive reaction and adding the incorrect amounts of reagents may cause poor results. Appropriately label your tubes with your group number and tube number!

2. Before mixing the reactants, you must calculate how much water must be added to make a total of 50 L (including the Taq Polymerase). This is necessary because the amount of plasmid that you add might differ from tube to tube.

3. If you add too much DNA, nonspecific amplification may occur—ask your TA how much DNA to add based on the approximate concentration of your plasmid samples.

4. Add all reactants, except the Taq, while the tubes are on ice. 5. Once all reactants (except Taq) are added to the tubes, spin them

briefly to bring all the liquid to the bottom of the tube. Note: Only spin tubes briefly (5 sec.), 0.2 ml tubes are thin-walled and can crack if microfuged for too long.

6. Lastly add the Taq polymerase.7. Keep the tubes on ice until the entire class is ready to load the

thermocycler.8. The thermocycler will run for approximately 3hrs. After the 3hr.

period is over, the thermocycler will stay at a constant 4C until the tubes can be placed in the refrigerator by the lab technician or TA. This will ensure that the PCR products will not degrade.

1.18 Examining the PCR product HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300 ml 1X TAE.2. Prepare a 2% agarose gel.3. Remove 20µL of PCR product from each tube; add to 4 µL of sample

buffer. Store the remaining PCR product at 4ºC. 4. DO NOT HEAT 100 BP MARKER!!!5. Load the PCR products onto gel along with 10 µL of 100 base pair

ladder.6. Run the gel for 1 hour at 100 volts.7. Stain, destain, and photograph the gel.

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1.19 Searches of the sequence using BLAST ( B asic L ocal A lignment S earch T ool) The plasmid DNA has been sequenced. You will receive a printout of the results. You will analyze this information using a computer program called BLAST.

To access the program, go to http://www.ncbi.nlm.nih.gov/BLAST

25

Module 2Gene Expression

Introduction In the following series of experiments, you will not be using the transformed cells you created in Module 1. K12 cells do not perform as well in expression experiments, so BL21 cells will be used in Module 2. BL21 cells express the GST protein much clearer. With these cells, you will induce the expression of the Glutathione-S-transferase (GST) and run a SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) to show that the protein was expressed. You will also perform a Western blot and use antibodies to confirm the presence of the GST.

2.1: Preparation of bacteria for SDS-PAGE (day before 2.2)

1. Streak an LB-amp plate with freshly transformed BL21+GST cells from your lab tech.

2. Streak an LB plate with control (non-transformed) BL21 cells from your lab tech.

2.2: SDS-PAGE preparation and Pierce Protein Assay

HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and

dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.

-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.

-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container.

-N-Butanol is flammable. Use caution.

Three hours before class …inoculate bacterial cultures: 1. Inoculate 2 x 2 ml of LB amp broth with transformed bacteria (1

colony each).2. To ONE of the LB amp cultures, add 60 L of 100mM IPTG.3. Inoculate 2 ml of LB broth with control (non-transformed) bacteria.4. Shake at 37C for 3 hours.

At lab time:

26

5. Obtain a large and small glass plate; wipe with methanol and a Kimwipe until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

6. Assemble the gel casting apparatus (See diagram in appendix). 7. Insert the comb and use a Sharpie to draw a line across the glass 1cm

below the comb. Once the line is drawn, remove the comb.8. Test to see if the apparatus is leak-proof. Squirt some water in

between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Placing several layers of paper towels on top of the rubber seal can do this. Make sure to remove the water before pouring the gel.

Before you pour your gel, have the TA or lab tech check your apparatus!

9. Prepare the resolving gel in a 15 ml tube according to the following directions. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL

Sterile dH2O 3.29 ml 4X resolving Buffer pH 8.9 2.60 ml

30% Acrylamide stock 4.00 ml TEMED 10 L

Last: Fresh 10% Ammonium persulfate 100 L

10. Gently swirl the solutions to mix WELL. 11. Using a Pasteur pipette, pour the gel by allowing the acrylamide

solution to run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

12. Overlay the acrylamide with N-Butanol to aid in the polymerization. Do this by gently adding the N-Butanol with a Pasteur pipette. You will be able to see a distinct line between the N-Butanol and the 4X Resolving solution.

13. Allow the gel to polymerize for AT LEAST 30 minutes (Any extra acrylamide mix in your tube will be a good gauge for polymerization. Make sure the cap is on).

14. While your gel is polymerizing, you can prepare your samples and standards for the Pierce Protein Assay if you have not done so already. Go to next section for instructions.

15. Once your gel has polymerized, pour off the N-Butanol into the sink and rinse with dH2O. Add a layer of dH2O on top of your gel using a Pasteur pipette.

16. Wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

Prepare the proteins for the gel and protein assay: 1. Split each culture into two 1.5 ml. You will have a total of 6 tubes (2 x

Transformed+IPTG, 2 x Transformed-IPTG, 2 x Untransformed).

27

2. Spin all tubes in the microcentrifuge for 1 minute.3. Decant the supernatant from each tube. 4. Resuspend one of each kind of pellet (i.e. transformed, transformed

plus IPTG and control) in 50 L lysis buffer. 5. Transfer this solution to its complementary tube. Do this for each

type of pellet. You will now have a total of 3 tubes.6. Sonicate each sample in ice, 3 times at 10-second intervals.

Sonicating breaks open the cells by sound waves. EAR PROTECTION REQUIRED!When sonicating, avoid touching the sides of the tube with the tip of the sonicator. The sample may become frothy; try to keep the sample from coming out of the tube. Turn off the sonicator and rinse the tip with dH2O in between samples and wipe with a Kimwipe.

7. Record the approximate total volume of each sample.8. Take 10 L samples from each sonicate; keep on ice for protein assay.9. Freeze the remaining samples at -20C. Label appropriately!

Pierce Protein Assay (See directions in Appendix G)Use the chart provided to develop a standard curve using BSA standards and to determine your own protein concentration. **NOTE: Do the entire PPA and standard curve development in DUPLICATE. Make two of every sample (label 1a, 1b, 2a, 2b, etc) and average the OD readings at the end for a more accurate standard curve.

1. Dilute protein sample: Your protein samples should be diluted 5:95. To do this, add 5 L of your sample and 95 L of sterile dH2O.

2. Make dye solution: Use Solutions A and B from the Pierce Protein Assay Kit. They should be mixed 50:1…but make up only the amount you will need (~40 ml). Mix the dye in a 50 ml tube.

3. Add BSA and dH20 according to the directions in Appendix G.4. Add 2ml of the dye to each one of your samples and standards.

Vortex.5. Incubate at 37C for 30 minutes. 6. Get OD values for standards and samples: TA will assist in the

operation of the spec. 7. Place your standard into a clean cuvette.

To clean the cuvette, rinse with dH2O. Make sure to dry the outside of the cuvette with a Kimwipe. Handle the cuvette only on the frosted sides.

8. Read OD at 562nm. 9. Repeat for each standard and sample, including duplicates.

If only using 1 cuvette, make sure to rinse with dH2O between each standard.10. Once the standards are complete, read your samples. You

should blank the instrument with dH2O and dye. If using only 1 cuvette, make sure to rinse with dH2O between each sample.

28

11. Find the average of each standard and sample duplicates.12. Establish a standard curve using the OD values obtained

with your BSA standards: graph OD (x-axis) vs. concentration (y-axis) on graph paper. Using this graph, calculate the protein concentrations in your three samples.

13. Use Excel to plot your data on a second chart (This is homework). Make sure to paste your Excel chart in your notebook properly labeled.

2. 3 SDS-PAGE HAZARDOUS CHEMICAL INFO:

-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.

-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.

-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container.

-Coomassie Blue Stain and Coomassie Blue Destain are highly flammable and irritating to the skin. WEAR GLOVES when handling and dispose of in appropriate waste container.

1. Remove the resolving gel from the cold room and pour a 4% stacking gel.

2. Mix the following components in a 15 ml tube. 3. Sterile dH2O 6.10 ml

4X stacking buffer pH 6.8 2.50 ml 30% Acrylamide stock 1.30 ml TEMED 10 L Last: Fresh 10% Ammonium persulfate 50 L

4. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and dry with a Kimwipe.

5. Add the ammonium persulfate to your tube. Mix gently.6. Pour the stacking gel as you did the resolving gel all the way to the

top of the small glass plate. If it overflows when inserting the comb this is okay. Clean the comb thoroughly with methanol before inserting.

7. Being careful to avoid making air bubbles, insert the clean comb until there is no air between the wells. This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

29

8. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while waiting.

9. Once the gel is set, remove them from the casting stand and assemble in the gel box. Note: Do not remove the comb yet.

10. Add ~115ml of PAGE Running buffer to the upper chamber. The buffer level should be half way between the big and small glass plate.

11. Add enough PAGE Running buffer to the lower chamber to cover the bottom half of the gels.

12. Carefully remove the comb.

Gel Set Up1. Calculate the volumes of sample, sample buffer, and water needed for

each tube (make 60 µl total so that even if some evaporates during boiling, there will still be 40 µl left):

-Each tube needs 60 µg of protein total. Using the concentrations you calculated off of your standard curve, calculate how many microliters equals 60 µg.-Each tube needs 1X sample buffer. You are given 6X sample buffer. Calculate how much 6X sample buffer is needed so that the final concentration is 1X.-Each tube needs a total volume of 60 µl. Figure out how much water should be added to each so that the total volume is 60 µl.

2. Place the remaining protein samples in the freezer.3. Boil samples for 5 minutes right before you are ready to load. 4. Load you samples into the gel in the following order (use gel loading

tips): Lane 1: 6X SDS sample buffer/Blank Lanes 2, 6, 10: Prestained protein marker (20 L per lane) Lanes 3, 7: Transformed Lanes 4, 8: Transformed plus IPTG Lanes 5, 9: Control (non- transformed)

5. Run the gel at 50mA for 1-1 ½ hours.6. Remove gel carefully from the gel apparatus. Use a razor blade to cut

the stacking gel portion away. Dispose of the stacking gel in the appropriate waste container.

7. Put the gel into a plastic container and cover with Coomassie stain. Microwave on low for 1 minute. Discard stain in appropriate waste container.

8. Transfer gel to destain. Wash and discard destain in correct waste container.

9. Submerge the gel in more destain. Shake gently overnight.

30

2.4 Dry Gel: Done for you

1. Discard destain in appropriate waste container.2. Rinse gel in dH2O water. Discard into appropriate waste container.3. Submerge your gel in more dH2O and place back on the shaker.4. Your TA/Lab tech will appropriately dry your gels using cellophane

and give to you during the next lab meeting.5. Make sure to observe and record gel appropriately.

2. 5 Pouring a Resolving Gel for SDS-PAGE and Western Blot HAZARDOUS CHEMICAL INFO:

-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.

-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.

-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container.

1. Pour a 12% polyacrylamide resolving gel:2. Obtain a large and small glass plate; wipe with methanol and a

Kimwipe until you hear a “squeaky” noise. Handle glass plates at edges.

3. Insert the comb and use a Sharpie to draw a line across the glass 1cm below the comb. Once the line is drawn, remove the comb.

4. Test to see if the apparatus is leak-proof. Squirt some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Placing several layers of paper towels on top of the rubber seal can do this. Make sure to remove the water before pouring the gel.

5. Before you pour your gel, have the TA or lab tech check your apparatus!

6. Prepare the resolving gel in a 15ml tube according to the following directions…BE SURE TO WEAR GLOVES…ACRYLAMIDE IS A NEUROTOXIN…DO NOT ADD THE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR GEL Sterile dH2O 3.29 ml 4X resolving Buffer pH 8.9 2.60 ml

30% Acrylamide stock 4.00 ml

TEMED 10 L

31

Last: Fresh 10% Ammonium persulfate 100 L7. Gently swirl the solutions to mix. 8. Using a Pasteur pipet, pour the gel by allowing the acrylamide

solution to run down along the side of the spacer. Add acrylamide until it is just barely above your Sharpie line. Try to avoid making bubbles.

9. Cover the acrylamide gel solution with N-Butanol. To do this, use a Pastuer pipet and gently place N-Butanol over the top of the gel. Once gel has polymerized, decant the N-Butanol and add a layer of dH2O. Wrap the gel apparatus in a damp paper towel and then Saran Wrap. Label appropriately. Store in cold room.

2. 6 SDS-PAGE gel and Western Blot

HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and

dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.

-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.

-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container.

-Western Blot Transfer Buffer is flammable. Wear gloves and use caution when handling.

1. Remove the resolving gel from the cold room and pour a 4% stacking gel.

2. Mix the following components in a 15ml tube. Sterile dH2O 6.10 ml 4X stacking buffer pH 6.8 2.50 ml 30% Acrylamide stock 1.30 ml TEMED 10 L Last: Fresh 10% Ammonium persulfate 50 L

3. Before adding the ammonium persulfate, remove the dH2O and dry with a Kimwipe.

4. Add the ammonium persulfate. Mix gently.5. Pour the stacking gel as you did the resolving gel all the way to the

top of the small glass plate. If it overflows when inserting the comb this is okay. Clean the comb thoroughly with methanol before inserting.

32

6. Being careful to avoid making air bubbles, insert the clean comb until there is no air between the wells. This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

7. Allow at least 30 minutes for the gel to polymerize. Thaw protein samples on ice while waiting.

8. Once the gel is set, remove them from the casting stand and assemble in the gel box. Note: Do not remove the comb yet.

9. Add ~115 ml of PAGE running buffer to the upper chamber. The buffer level should be half way between the big and small glass plate.

10. Add enough PAGE running buffer to the lower chamber to cover the bottom half of the gels.

11. Carefully remove the comb.

Gel Set Up:1. Calculate the volumes of sample, sample buffer, and water needed for

each tube. This should be the same as in 2.3.2. Place the remaining protein samples in the freezer.3. Add 6X SDS sample buffer to each protein sample.4. Boil samples for 5 minutes right before you are ready to load.5. Load you samples into the gel in the following order:

Lane 1: 6X SDS sample buffer/Blank Lanes 2, 6, 10: Prestained protein marker (10 L per lane) Lanes 3, 7: Transformed Lanes 4, 8: Transformed plus IPTG Lanes 5, 9: Control (non- transformed)

6. Run the gel at 50mA for 1-1 ½ hours and then set up Western blot.

Blotting ProcedureThe transfer will be accomplished using the Hoeffer Semi Dry Transfer Apparatus. This unit transfers proteins from a polyacrylamide gel to a nitrocellulose membrane by means of a low current and low voltage transfer.

1. Rinse the anode and cathode of the transfer apparatus with dH2O. Be careful not to get the leads or interlock housing wet.

2. Prepare the gel for transfer. Carefully cut away stacking gel with a razor blade. Measure the gel and record the dimensions.

3. Cut a hole in a Mylar mask 2 mm smaller than the gel. Center the mask on the anode of the transfer apparatus.

4. Cut six pieces of blotting paper and one piece of nitrocellulose membrane the same size as the gel. Measure and cut carefully; they must not be larger than the gel! Make sure to notch the nitrocellulose for orientation purposes!

5. Soak the blotting paper in Western blot transfer buffer.

33

6. Rinse the nitrocellulose membrane with dH2O, then soak it in Western blot transfer buffer for 5 minutes.

7. Put one piece of the blotting paper over the opening in the Mylar mask. Roll a test tube over the paper 3-4 times to push all air bubbles out. You will need to use moderate pressure to be effective.

8. Repeat this process adding two more blotting paper layers.9. Add the nitrocellulose paper to the stack and roll out the air bubbles.10. Add the gel. Do not roll. Be careful setting the gel on the

stack. Try to line it up correctly the first time as some proteins may stick to the membrane on contact and moving the gel around will affect the quality of your blot.

11. Add the last three strips of blotting paper, one at a time, carefully rolling out the bubbles each time.

**NOTE: If it is too hard to place the gel exactly on top of the nitrocellulose membrane, you can put the gel down first and then the nitrocellulose, but you need to remember to FLIP THE STACK after you have finished putting the filter paper on top so that the nitrocellulose is BELOW the gel.**12. Put the top on the transfer unit. Set a flask with 1L of water on

top to add pressure.13. Connect the short safety interlock lead on the cover to the jack

on the base. Plug the leads into the power supply.14. Turn on the power (0.8 mA per cm2 of gel surface). Transfer for

1½ hour.15. Turn off the power supply. Disconnect the leads and safety

interlock.16. Use forceps to remove the nitrocellulose membrane. Place it

face up on fresh blotting paper. Record the orientation of the lanes, and then wrap the nitrocellulose in plastic wrap and store at -20C. Blotting papers and gel can be thrown away.

2.7 Primary Antibody Blocking (day before 2.8)

1. Cut the nitrocellulose through the center lane of the prestained marker. Wrap one half of the blot in Saran wrap, label, and place back into the freezer.

2. Submerge the other half of the blot with 25ml of blocking solution. Rock at room temperature for 1 hour.

3. Decant the blocking solution into the sink. 4. Wash the blot with 50ml TBS. Repeat.5. Add the primary antibody (Anti-GST produced in rabbits diluted

1:5000 in 10 ml of solution containing TBS, 0.1% Tween-20, and 1% dry non-fat milk). Add enough to submerge the nitrocellulose filter.

6. Rock in the cold room overnight.

34

2.8 Staining and Detection of Western Blot

HAZARDOUS CHEMICAL INFO:-Amido Black Stain and Destain are flammable irritants. Wear

gloves when handling and dispose of in proper waste containers.

Staining with Amido Black:1. Remove nitrocellulose blot from the freezer and cover with a minimal

amount of amido black.2. Rock at room temperature for 5 minutes.3. Decant stain back into its original container then wash the blot with

amido black destain until all background color is gone.4. Decant the destain into the proper waste container.5. Place the blot on filter paper to air dry.6. Observe the stained blot. Save it to compare to the immunostained

half. Wrap the blot in plastic wrap. Copies of the stained blot should be made for each group member. Scanning and printing the blot can achieve this.

Detection of the antibody:1. Decant the primary antibody into the sink.2. Briefly wash the blot with TBS-T. Decant the TBS-T into the sink.

Repeat 3X. 3. Add the secondary antibody (Goat anti-rabbit IgG alkaline

phosphatase conjugated diluted 1:10000 in TBS-T). Save a small volume of the secondary antibody in a 1.5 ml tube to use as a control, keep on ice.

4. Rock the blot at room temperature for 1 hour. 5. Decant the secondary antibody into the sink. 6. Wash the blot with 50 ml of TBS-T. Rock at room temperature for 5

minutes.7. After 5 minutes, decant the TBS-T into the sink, add fresh TBS-T and

repeat for a total of (6) 5 minute TBS-T washings.8. Add 500 L of the NBT-BCIP to the secondary antibody you saved. A

blue color should develop within a couple minutes.9. Add 10 ml NBT-BCIP solution directly to your blot. Agitate until color

develops.10. Once color has developed, decant the NBT-BCIP solution into its

appropriate waste container. Rinse the nitrocellulose with dH2O. Decant into the sink.

11. Air dry the nitrocellulose on filter paper. Observe and record. Wrap the nitrocellulose in plastic wrap and store in your notebook. Copies of the blot should be made for each group member.

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Module 3Screening a Plasmid Library Through Protein Expression

Introduction In order to clone genes, scientists often turn to huge libraries of plasmids that have a variety of sequences inserted into them, one sequence per plasmid. The trick is to pick out the one that carries your gene. To find the gene, we can use a plasmid that has a promoter before the insert and that expresses the mRNA for the inserted genes. The mRNA is translated into protein. In order to find the bacteria expressing you gene, you can use antibodies against the protein gene product. This experiment is designed to screen a library for the GST or GFP gene (your TA will inform you of the choice).

You will grow the library bacteria on agar and screen the clones for the ones expressing GST. Expression by individual clones is detected by overlaying the plates with nitrocellulose filters that have been soaked with isopropylthio--galactoside (IPTG) to induce protein expression. The filter is then incubated with anti-GST antibodies and the GST producing colonies identified. The GST producing clone is then isolated and a pure culture grown. The plasmid is isolated and a restriction enzyme digest is done to cut out the receptor gene insert. The products from the digest are run on an agarose gel for analysis.

3.1 Plating the bacteria _

1. Serially dilute an E. coli culture that is the library with sterile dH2O so that at least one plate has between 100 and 200 colonies. Use four tubes with the following amounts of dH2O: 100 L, 150 L, 200 L, 250 L. Ask your TA how much library to add.

2. Plate 100 L of your diluted bacteria on four properly labeled LB amp plates.

3. Incubate plates upside down overnight at 37C.

3.2 Overlaying the Plates

1. Pre-soak two nitrocellulose membranes in 10 mM IPTG and air-dry before use. Gently handle the NC Paper with forceps. Soak the membranes separately. Dispose of the IPTG in the appropriate waste container.

2. Carefully overlay the plates with the nitrocellulose filter. 3. Incubate at 37C for 3 hours right side up. 4. Using a needle and India ink, mark the filters. Choose an asymmetric

pattern so you will be able to match the filter and the plate later. Make the mark at the edge of the plate. Make sure the ink marks the plate as well as the filter.

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5. Remove the nitrocellulose filter and soak in ~7 ml of TBST. Wrap in plastic wrap, label and store in refrigerator/cold room. Store LB amp plates in refrigerator/cold room.

3. 3 Identifying positive colonies

HAZARDOUS WASTE INFO:-NBT/BCIP is extremely toxic. WEAR GLOVES when handling and

dispose of all liquid waste containing NBT/BCIP in proper waste container.

1. Incubate the nitrocellulose filters in 10 ml of blocking buffer in Petri dishes. Rock at room temperature for 1 hour. Wash filters in separate containers.

2. Decant the blocking buffer and wash the filters with 6-10 ml TBST.3. Decant the TBST and add 10 ml of primary antibody (Anti-GST

produced in rabbits, diluted 1:5000 in a solution containing TBS, 0.1% Tween, 1% dry non-fat milk). Rock at room temperature for 1 hour. When done, decant and save the antibody in the refrigerator.

4. Wash the nitrocellulose filter for 5 minutes in 6-10 ml TBST; rock at room temperature. Repeat for a total of three washes.

5. Incubate the nitrocellulose filters in 10 ml of secondary antibody (Goat anti-rabbit IgG diluted 1:10000 in TBS-T). Rock at room temperature for 45 minutes. When finished, save the secondary antibody for a check below.

6. Wash the nitrocellulose filter for 5 minutes in 6-10 ml of TBST; rock at room temperature. Repeat for a total of three washes.

7. Decant the TBST and add 6 ml of the NBT/BCIP solution to the filter. Add 500 L of NBT/BCIP to the secondary antibody you saved as a control to check for color development (a blue color should develop).

8. Once color has developed on the nitrocellulose filter, decant the NBT/BCIP in the appropriate waste container and soak in stop solution for 10 minutes. Air-dry the filter. Wrap the filters in plastic wrap and store in the cold room.

Wednesday 3.4 Isolation of positive colonies

1. Note positive colonies on the NC and find the corresponding colonies on the agar plate.

2. Use a Pasteur pipet (or sterile toothpick) to select four positive colonies and 1-2 negative colonies for control

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3. Put the agar plug in a tube containing 10ml of LB amp broth. Incubate overnight in a 37C shaking waterbath.

3.5 Mini prep plasmid isolation Note: Appropriately label all tubes and matching columns so there is no confusion!

Part I. Preparing the lysate:1. Transfer 1.5 ml of bacterial culture into 2 microfuge tubes; do this for

each of your tubes.2. Centrifuge at 12,000 rpm for 3 minutes at 4C.3. Decant as much as the supernatant as possible. Resuspend the pellet

in 200 L cell resuspension solution. Transfer this mix to the matching tube and resuspend the second pellet.

4. Add 200 L cell lysis solution and invert 4 times to mix.5. Add 200 L neutralization solution and invert 4 times to mix.6. Centrifuge lysate for 5 minutes at 13,000 rpm.

Part II. Plasmid DNA Purification:1. Obtain 1 mini-column and 1 syringe for each culture. Remove the

plunger from the syringe and attach the column to the syringe. Place the column in a small test tube.

2. Shake the resin bottle to resuspend the resin then add 1 ml to each mini-column/syringe assembly.

3. Carefully transfer the cleared lysate from #6 above to the resin.4. Insert the plunger and push the resin/lysate into the minicolumn.

Part III. Washing:1. Detach the mini-column from the syringe. Remove the plunger from

the barrel. Reattach the mini-column.2. Add 2 ml column wash solution. Insert the plunger and push the

column wash through the mini-column.3. Remove the syringe and transfer the mini-column to a 1.5 ml

microcentrifuge tube. Centrifuge at 13,000 rpm for 2 minutes.

Part IV. Elution:1. Transfer the mini-column to a new microcentrifuge tube.2. Add 50L sterile dH2O directly into the column and wait 1 minute.3. Centrifuge at 13,000 rpm for 20 seconds.4. Remove and discard mini-column. Store DNA at -20C. Label tubes

appropriately!

3.6________________________ 1. Remove one DNA sample from freezer and thaw on ice.2. Transfer 18 L of the DNA sample to a clean microcentrifuge tube.3. Add 2 L of 10X React 3 Enzyme buffer to the tube.

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4. Add 1 L of BamH1 and 1 µL of EcoRV to the tube. Mix with pipette man. Enzymes need to be kept on ice. Enzymes should be added last to well mixed solutions.

5. Incubate at 37C for 45 minutes.6. Microfuge at 12,000 rpm for 5 seconds.7. Add 5 L of sample buffer to the digests.8. Remove 10 L of the corresponding non-digested plasmid DNA to a

clean microfuge tube. Add 4 L of sample buffer and 10L TE. 9. Load onto a 0.7% agarose gel:

Lane 1: Hind III marker. Heat at 65C for 7 minutes before loading onto gel! Lane 2: Non-digested plasmid Lane 3: Digested plasmid

9. Run at 100 v for approximately 1 hour.10. Stain the gel in ethidium bromide, destain and photograph.

Record and interpret results.

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Module 4Human Forensic DNA Analysis

Introduction

This module allows you to analyze some of your own DNA for markers that are used in forensic analysis and also to simulate the analysis of criminal evidence. One marker that you will use is the TPA-25 sequence from an intron of the tissue plasminogen activator gene. This is a member of the Alu family of insertions in our genomes. You will amplify regions of chromosome 8 to determine whether the TPA-25 insert is in the intron. We can screen for this insertion sequence using polymerase chain reaction (PCR) because it has no phenotypic consequence, i.e. no health or other problems arise from its presence.

We will also amplify a region of chromosome 1 region D1S80 to look for a polymorphic sequence, that is a region that has a variable number of repeats (VNTR). Again, we will use PCR to amplify a region and compare results among lab members to see how we can identify each one uniquely by the size (14-40 repeats) of the PCR product from this region. The sequence we will amplify is in a non-coding region and has no phenotypic consequences.

The DNA template for your PCR will be your own, from a swab of your mouth.

The following primers are used to bracket the TPA-25 locus- The forward primer:

5’-GTAAGAGTTCCGTAACAGGACAGCT-3’

The reverse primer:

5’-CCCCACCCTAGGAGAACTTCTCTTT-3’

The following primers are used for the D1S80 VNTR-

The forward primer:

5’-GAAACTGGCCTCCAAACACTGCCCGCCG-3’

The reverse primer:

5’-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3’

It is important for you to pipet accurately and to carry out these experiments carefully or your results will not be reproducible and definitive – not very good evidence for a crime scene investigation.

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4.1 Part A: To Make Template DNA:

1. You will be assigned a number by the TA. This is your sample identification for this lab.

2. Label a 50ml tube containing 0.9% saline with your number.3. Pour all of the saline solution into your mouth and swish vigorously

for 60 seconds or more. 4. Return saline to the tube and replace cap.5. Load your tube into the IEC Centra and centrifuge at 2,800 rpm for 10

minutes. 6. Carefully decant the supernatant into the sink; do not disturb the

pellet. Place the tube on ice.7. Resuspend the 10% Chelex solution and add 500 L to the pellet. Note: When dealing with Chelex make sure to use the wide-mouth pipette tips.8. Transfer 500 L of the resuspended Chelex/cell solution to a sterile

1.5ml microfuge tube labeled with your name. 9. Incubate your sample in a boiling water bath for 10 minutes.10. Remove your sample from the water bath and cool on ice for ~1

minute.11. Spin your sample in the microcentrifuge for 30 seconds to pellet

the Chelex beads.12. Transfer 200 L of the supernatant to a clean, sterile 1.5 ml

tube labeled with your number and place it on ice. Do not transfer any of the Chelex.

13. Waste is biohazardous! Discard appropriately.

Part B: Set Up the PCR:1. Label the cap of a 0.5 ml PCR tube with your initials.2. Use the amplification chart on the following page as a checklist while

adding reagents to the 0.5 ml PCR tube. Do not cross contaminate! Note: Keep all your tubes on ice while adding materials to them. Keep them on ice until you are ready to load into the thermocycler.

3. Add the following reagents accurately:

Reagents (uL)Tube 1 TPA-25

Tube 2 TPA-25 Tube 3 D1S80 Tube 4 D1S80

Template DNA 5 5 5 510X PCR buffer 5 5 5 525 mM MgCl2 5 5 5 5

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1.25 mM dNTPs 8 8 8 8Forward Primer 1 XXX 1 1Reverse Primer 1 1 1 XXXTaq polymerase 0.5 0.5 0.5 0.5Sterile dH2O 24.5 24.5 25.5 25.5(total volume/tube= 50 ul)

Make sure you put the correct primers in their corresponding tubes! You will be given 4 different primers: Forward & Reverse for D1S80 and Forward & Reverse for TPA-25.

4. Cap your tubes and place them in the thermocycler. Select the appropriate protocol for each set of primers.

5. After preparing the tubes for the PCR, store your remaining DNA in a labeled tube at -20C.

TPA-25 PCR Conditions

94C 5 min Initial Denature94C 1 min

30X56C 1 min72C 1 min72C 10 min Final

Elongation4C HOLD ****************

D1S80 PCR Conditions

94C 5 min Initial Denature

94C 1 min5X63C 1 min

72C 1 min

94C 1 min

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25X64C 1 min72C 1 min

72C 10 min Final Elongation

4C HOLD ****************

4.2 Preparation of 2% Agarose Gel WEAR GLOVES WHEN PREPARING AND RUNNING GELS!

1. Prepare a 2% agarose gel.2. Microwave for ~1.5 minutes to dissolve the agarose.3. Let the agarose cool slightly before pouring, ~2 minutes. Note: The

2% agarose solution will solidify quickly! Pour gel while still relatively hot.

4. Pour the agarose into the gel-casting tray. 5. While the gel is setting, prepare your DNA samples.6. In a 0.5ml tube, mix 4µL sample buffer with 20µL unamplified DNA.

Repeat with each PCR product.7. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be

loaded directly on the gel. 8. Once your gel is set, remove the comb and place the gel in the

running box. 9. Cover the gel with TAE buffer and load your DNA samples.10. Run gel at 120 volts for 45 minutes to 1 hour.11. Once the electrophoresis is complete, stain your gel for ~10

minutes in ethidium bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.

12. Examine, photograph, and destain gel.

4.3 Interpreting your results_____________________________________Examine your results and consult additional literature. Draw preliminary conclusions. Your TA will ask you to describe and explain your gel.

4.4 to 4.5 Repeating PCR When dealing with forensic science, tests need to be repeated to make sure the results are the same and therefore correct. In 4.1 and 4.2, you isolated your own DNA, used PCR to amplify it, and ran it on a gel. Now, you will

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use the remaining isolated DNA and repeat PCR using varying amounts of water, MgCl2, dNTPs, primers, template DNA, 10X PCR buffer, and Taq. Use your past knowledge and experience of PCR to calculate the amounts and concentrations of the PCR reagents to use.

4.6 to 4.7 Murder Investigation You are a lab technician working at a forensics lab. A DNA sample has come in. The sample is evidence that has come from a murder scene and the murderer is unknown. The DNA sample was collected from a bottle found at the crime scene. The investigators have swabbed the bottle and placed the DNA in a sterile saline solution to preserve it until processing. Investigators have also collected DNA samples from 4 murder suspects and from the murder victim. You must process and compare the DNA sample found at the crime scene to those collected from the murder suspects and the victim. From your results, determine who is guilty of the crime and prove why they are guilty.

In addition, other forensics labs are processing the same evidence/samples. Your results should match up with all other labs if they are correct. If your results do not match up with the other forensics labs’ results, trouble shoot and repeat until all labs have the same results. Follow the protocols that you have just used with your own DNA to prepare the DNA samples for PCR.

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Module 5 Microarray Technology

IntroductionModified from Microarray Technology Lab Manual by Ahmad Chaudhry, et al. Vermont Genetics

Network, Microarray Outreach Program, University of Vermont, Spring 2004.

In this module, students will learn how gene expression in yeast (Schizosaccharomyces pombe) is changed after exposure to oxidative stress caused by the addition of hydrogen peroxide (H2O2), an oxidizing agent. Experimentally, the yeast will be grown for 48 hours in 1/2x YPD broth containing 3x glucose. The culture will be split into a control and treated group. The control will be exposed to Hanks Buffered Saline Salt (HBSS) only, while the treated yeast are exposed to 0.5mM H2O2 for 1 hour. RNA will be extracted and prepared for use on Affymetrix® GeneChips. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the yeast GeneChips. The resulting gene expression data will be uploaded into special bioinformatic data analysis software where students are taken through complex data analysis procedures.

Days One:

Today we will be treating the yeast Schizosaccharomyces pombe (a haploid fission yeast) [NRRL Y-128 or ATCC38366] with hydrogen peroxide [H2O2] to understand the gene expression effects during oxidative stress conditions in this simple eukaryote. The H2O2 treated yeast, and an untreated control will be lysed using a combined procedure employing a lyticase enzyme and a buffer containing guanidinium isothiocyanate after the 1-hour treatment procedure. The first step uses lyticase which degrades the poly--1,3 glucan bonds in the cell wall of fungi, resulting in the formation of spheroplasts (a fungal cell without a cell wall). The second step uses RLT buffer (guanidinium isothiocyanate) that ruptures the cell membrane thereby allowing recovery of total RNA on a common silica gel spin column manufactured by Qiagen.

The Silica Column ChemistryGuanidium isothiocyanate (GITC or GSCN) is a chaotropic salt at about 5 M in the RLT buffer. This solution at a reduced pH (6.4) and used in conjunction with ethanol and water disrupts the molecular characteristics of water and forces the RNA to be less soluble. This promotes nucleic acids to electrostatically bind to the silica. A stoichiometric balance of GITC, ethanol, pH, and water controls the binding of either RNA or DNA to the silica. Therefore, changes in this stoichiometry will select a majority of either one or the other. This not a 100% selective procedure and DNase treatment is required. The use of ethanol in rinse RPE buffer maintains the bond between the silica and RNA. The recovery of RNA off the membrane

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is accomplished with water because RNA is very soluble in water and can no longer maintain an electrostatic bond with the silica once the water structure has been re-established.

Day Two:We will be preparing the mRNA for microarray target preparation and then, cDNA synthesis.

Technical Overview:Today you will be starting to prepare the target for the Genechip analysis. This is a lengthy procedure and will be broken down over several lab periods. In short, you will be starting with mRNA, which comprises 0.5-2% of the total RNA in typical eukaryotic cells. First, you will generate the first complementary DNA (cDNA) strand from the mRNA. This is called the first strand cDNA synthesis. This step utilizes a primer with a sequence of 24 T's in a row and is called oligo d(T), which binds to the poly A tail of eukaryotic mRNA. The primer also contains the consensus-binding site for T7 RNA polymerase (T7 promoter sequence). Once the primers have been hybridized to the poly-A mRNA, reverse transcriptase is added and the first strand of cDNA is synthesized. It is important to note that the 5s, 5.8s, 18s, and 25s rRNA do not have a poly-A tail and will not be primed or synthesized in this procedure.

The sequence of T7 Oligo d(T)24 is:

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The next step in the cDNA reaction is the synthesis of the second cDNA strand from the first cDNA strand. This is done by adding RNase H, DNA polymerase I, and DNA ligase. The RNase H causes many strand nicks in the phosphodiester bonds of the bound mRNA strand creating the availability of 3'OH groups. DNA polymerase uses these 3’ OH groups as a priming site; thus synthesizing another cDNA strand until it reaches another nicked site. The DNA polymerase can only displace RNA, not DNA. After cDNA has been synthesized from all the nicks, the areas between the nicks need to be filled in or ligated. This is accomplished using DNA ligase. After the two hour synthesis process, all the RNA has been replaced with cDNA and ligated to form one large cDNA strand. However, because the DNA polymerase does not synthesize completely to the end of the strand, a T4 DNA polymerase is added. This ensures the synthesis of the T7 promoter site from the original T7 oligo d(T) primer used in the first synthesis reaction.

Synthesis of cDNA:The starting material for your target preparation will be high quality, fully intact, total RNA from yeast. The minimum concentration to start with is 100-270 ng/μl. After isolation and purification of the yeast RNA, the concentration is determined by absorbance at 260 nm on a spectrophotometer (1 O.D.= 40 µg/mL RNA). The A260/A280 ratio should be approximately 1.8-2.1. We are checking the quality of the RNA by running an aliquot on the Agilent Bioanalyzer 2100 (performed prior to class at UVM core) and running on an agarose gel (EGEL) prior to starting the assay. More appropriately, a denaturing formaldehyde gel would be used, but we will not have time to perform this.

Overview of cDNA Synthesis Reactions

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Day Three: We will be cleaning the cDNA and preparing it for an in Vitro Transcription to synthesize an antisense cRNA.

Cleaning the cDNA:After synthesis of the cDNA, it will need to be purified before proceeding to the next step. This will be accomplished using a solution of phenol-chloroform-isoamyl alcohol (PCI) at a specific pH. The underlying principle is that both phenol and chloroform cause proteins/enzymes to become denatured and subsequently soluble in the organic phase, while the cDNA will remain in the aqueous phase. This is performed by mixing the cDNA sample with PCI and transferring the full volume to a phase lock gel tube. The phase lock gel tube is centrifuged allowing separation of the aqueous and organic phases with a gel barrier. This allows for easy recovery of the cDNA in the aqueous phase. It is important to note that the pH of the PCI is important because this will determine which layer the cDNA will be retained in (the aqueous or organic phase). To ensure the cDNA is in the aqueous phase, the pH of the PCI needs to be basic, which is accomplished through a layer of TRIS pH 8.3 over the PCI solution.

The aqueous phase is further purified through a precipitation step using ethanol [an alcohol] and ammonium acetate [a salt]. Because DNA is negatively charged, salt is used to mask the charge, causing it to precipitate. The ethanol is used in conjunction with the salt because it is non-polar and DNA will not readily dissolve in it. This reaction is sometimes done at -20°C to encourage maximum DNA precipitation. After the precipitation, the cDNA is centrifuged to a pellet with a visualization agent called Pellet Paint, an additive that contains glycogen (a co-precipitate) and a dye. This helps form a high quality visible pellet on the bottom of the tube. The pellet is then washed several times with 80% ethanol, dried, and resuspended in DEPC water.

The In vitro Transcription (IVT):The IVT step uses the T7 promoter site that is attached to the T7 Oligo d(T)24 primer used in the first strand cDNA synthesis. A T7 RNA polymerase binds to this site and copies the opposing cDNA strand, synthesizing an antisense cRNA using the two standard nucleotides, A and G and two modified nucleotides; biotinylated uracil and cytosine. This generates a complementary cRNA [antisense] strand to the original mRNA with biotinylated nucleotides. These biotinylated nucleotides will be needed in the next step for staining of the RNA using strepavidin phycoerytherin.

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Day Four:We will be cleaning the cRNA before fragmentation. It is important to have “clean” cRNA before applying to the chip to reduce non- specific binding, as well as being sure that the cRNA has fragmented.

Cleaning and fragmenting the biotinylated cRNA:Using the Qiagen RNeasy system, the biotinylated cRNA must be purified. The cRNA needs to be free of enzymes, dNTP's, reducing agents such as DTT [dithiothreitol] and resuspended in DEPC water. The clean cRNA can then only be accurately quantified. Remember that enzymes and dNTP's also have an absorbance at 260 and/or 280nm that will affect the quantitation of the molecμle of interest. That is why [for instance] you cannot quantify a raw PCR reaction before and after thermocycling, because all components are still there, just assembled differently.The cleaning is performed by adding beta-mercaptoethanol, ethanol, and a buffer to the cRNA mix to reduce the proteins. These reagents aid in the solubilization of proteins and prepares the cRNA for binding to a silica-based spin column (pink basket of the RNeasy kit). The cRNA is washed and eluted using DEPC water. The cRNA is then quantified using a spectrophotometer at 260nm for nucleic acids and 280nm for proteins. A 260/280nm ratio of 1.8-2.1 indicates a clean cRNA sample that can be used for subsequent steps. 260/280 ratios of less than 1.8 indicate that the cRNA is contaminated with too much protein and should be purified again.

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Performed by UVM:Once the cRNA is cleaned and quantified, it will be given to

UVM’s Micoarray Core Lab to perform the following last steps. The hybridization mixture contains several controls that provide information about the success of the hybridization procedure. These controls are the B2 oligo for which there are several hundred probes along the outer edge of all expression arrays and checkerboard pattern in each corner. These predefined patterns provide signals for the Affymetrix Microarray Suite software to perform automatic grid alignment during image analysis. They can also be used to align the grid manually. The fluorescence intensities for control oligo B2 are not used for analyzing data. The bioB, bioC and bioD are biotinylated gene fragements much like your sample, which represent the biotin synthesis pathway from the bacteria E. coli. The Cre control is the recombinase gene from P1 bacteriaphage. These biological controls are prepared at standardized concentrations and signal intensities obtained on these genes provide information on how well the hybridization, washing and staining procedures have performed.

After the hybridization mixture has been made, it is denatured at 94C for 5 minutes and injected into the GeneChip through the injection ports on the back of the chip (See diagram in appendix). The GeneChip is then placed in a rotating incubator and the biotinylated target that you prepared is allowed to bind (hybridize) to the sequence specific probes on the array.

The staining procedure involves using a fluidic station designed to accommodate GeneChips. Two solutions are prepared: a strepavidin-phyoerytherin dye (SAPE) and a biotinylated anti-phycoerytherin antibody (BAP). Before placing the GeneChip into the fluidic station, the fragmented cRNA hybridization mixture is manually removed and replaced with buffer.

The chip is placed in the fluidics station and an automatic process of washing and staining occurs for approximately 2 hours. The procedure starts by first staining with SAPE. The SAPE will bind the biotinylated cRNA that is hybridized to the probes on the chip. It is washed and then stained with BAP. BAP will bind to the SAPE. The chip is washed again and another SAPE staining is carried out to bind to the BAP. The double staining procedure is employed to amplify the signal intensities. This allows for the detection of low transcript mRNA’s that might otherwise not be detected. After staining, the sample is washed and ready for scanning.

The GeneChip is scanned after the washing and staining protocols are complete. The chip is placed in the scanner that is equipped with an Argon 488nm laser and the beam is scanned back and forth across the glass surface at a resolution of 5m. Fluorescence is detected using a high-resolution photo multiplier tube

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(PMT). This enables high-resolution detection of fluorescence from the probe set feature (pixel), which is only 19m square. There are ~1 million probe areas on each GeneChip!

After collecting and analyzing the image data, the results are archived and burned onto a CD. Because there is so much data, it is necessary to analyze it using a special software package such as GeneSifter. This software can handle tens of thousands of data points at one time and data analysis can often take months. Therefore, it is critical that the data meet specific control specifications before this time consuming analysis is performed.

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Image from http://www.microarray.lu/en/MICROARRAY_Overview.shtml Microarray Center CRP

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TO CREATE AN RNase-FREE ZONE:**It is very important that you follow these directions exactly—minimizing contamination and degradation of your samples is crucial to the success of the experiment**

1. Tape off an area with labeling tape. Label the area as an “RNase-free zone”.

2. Wearing gloves, wipe down the area with ethanol, then with the RNase Away® solution.

3. Wipe down the pipette men you will use during your experiment with the RNase Away® solution.

4. Once the pipette men are cleaned, place them in the RNase-free zone.

5. Wipe down any instruments or bottles that you will come in contact with (racks, tube holders, etc.) with ethanol. Place them in the RNase-free zone. Make sure to read through the procedure and plan accordingly.

6. Follow the guidelines bellow to insure an RNase-free zone:7. If you are to touch any part of your body or hair with your

gloved hands, change your gloves. Your skin and hair naturally contain DNase and RNase to battle against everyday bacteria and germs.

8. If you walk to another area in the lab, change your gloves before working in the RNase-free zone again.

9. If you touch a piece of equipment (microcentrifuge) or lab bench that has not been wiped down, change your gloves before working in the RNase-free zone again.

10. Use only unopened sterilized tips, tubes, etc. If you are unsure about the “freshness”, ask your Lab Tech.

11. Use only sterile distilled water.12. Work quickly, but carefully. RNA degrades quickly.

Day Before 5.1: Preparation of Yeast Cultures (Done for you) Using sterile technique, 500mL of YPD (Yeast Peptone Dextrose) broth will be inoculated with an isolated yeast colony.The culture will stir on a stir plate at room temperature (22-25C) for 48 hours.

5.1 Treatment of Control and Treated Yeast Cultures (DAY 1) 1.5 Hours Before Lab (Done for you):

1. From the 48 hour old culture, aseptically transfer 40.0 ml of the broth culture into two (2) sterile 125 ml flasks each containing a stir bar using a sterile 50 ml pipet.

2. Label one flask as “treated” and aseptically add enough H2O2

from the “working stock" to achieve a final H2O2 of 0.5mM [See

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below]. Label the control flask as “control” and add an equal amount of HBSS as you did H2O2.

Preparing and Determining the Concentration of H2O2a. Preparation of H2O2 Stock Solution: Combine 5.0μL of

30% H2O2 and 495 μL of Hanks Balanced Saline Salt [HB without phenol red, Mg, or Ca. Vortex.

b. Working H2O2 solution : Make a 1:10 dilution by combining 100μL H2O2 stock solution with 900μL HBSS and vortex. Use this for spectrophotometer measurement and for the experiment.

c. Blank the spectrophotometer with HBSSd. Measure the absorbance at 240nm.e. Concentration of H2O2 in Working Solution : (Ab240) x

229 = ____mM of H2O2 [B].f. Example: H2O2 to add to 40 ml flask= A/B x 40ml x1000

= ul of working solution to use in experimentWhere A=0.5mM B=your concentration in working solution

3. Place each flask on separate stir plates and stir at room temperature for 1 hour at [as close as] the same speed as possible. Cultures should be ready for harvest 30 min after the start of the first class period. Therefore, H2O2 is added 30 minutes before the start of the class.

During Lab:1. Prepare an RNase-free work zone according to the directions

above.. 2. Prepare fresh lytic enzyme at 10 Units/μl by adding 1 ml of

DEPC water directly to the lyticase vial. a. 10,000 U Lyticaseb. 1 ml DEPC Water

3. Vortex well and invert several times to insure complete mixing.4. Transfer to a 1.5mL microcentrifuge tube. This solution is

stable for 12 hours.5. Prepare fresh DNase I solution using the Qiagen DNase kit and

store on ice.6. Add 10L DNase I and 70L RDD Buffer to a 1.5mL

microcentrifuge tube7. Transfer 1.5mL of the Control yeast culture to a 1.5mL

microcentrifuge tube.8. Transfer 1.5mL of the Treated yeast culture to a 1.5mL

microcentrifuge tube.***Perform the following steps 9 through 39 to each yeast culture!

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9. Spin down 1.5 ml of yeast culture at 8000x g for 2 minutes using an Axygen 1.7 ml tube in a microcentrifuge at room temperature.

10. Carefully remove supernatant (without disturbing pellet) using a P1000 micropipet, discard supernatant

11. Add 1 ml of sterile DEPC water, vortex, and spin again at 8000 x g for 2 minutes.

12. Remove and discard supernatant. Remove as much of the liquid as possible from the yeast pellet. You may need to use a smaller pipet to get any remaining liquid.

13. Add the following to the yeast pellet anda. SG buffer 100 μl b. Lyticase solution (10U/μl) 30 μl

14.Incubate for 30 min at room temp (22-25°C), gently swirl tube every 10 minutes to generate spheroplasts. Spheroplasts must be handled gently.

15. Prepare RLT buffer (from Qiagen RNeasy Kit) for the nest step by adding 10ul of β- mercaptonethanol (BME) to 1ml RLT buffer. USE BME under fume hood! This buffer will now be called b-RLT.

16. After 30 minute incubation, add 350 µl b-RLT buffer to lyse spheroplasts.

17. Vortex vigorously for 1 minute. Ensure your tube is tightly capped by holding lid closed while vortexing.

18. Add 250 µl of 100% ethanol to the tube and briefly vortex. Do not centrifuge. A precipitate may form after the addition of ethanol, but this will not affect the RNeasy procedure.

19. Apply the entire sample to an RNeasy mini column. 20. Close the tube gently and centrifuge for 15 sec at full

speed. 21. Discard the flow through tube and place the RNeasy

column into a new 2 ml capture tube. 22. Add 350 µl RW1 buffer to the RNeasy column to wash

the column. Close the tube gently and Centrifuge for 15 sec at full speed.

23. Applycolumn membrane. Incubate at room temp for 15 min.

24. Transfer RNeasy column to a new 2 ml capture tube.25. After the 15 minute incubation, a

RNeasy column and spin at full speed for 15 seconds.26. D

a new 2 ml capture tube.27. Pipet

column. Close the tube gently and centrifuge for 15 seconds at full speed.

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28. Discard the flow through tube and place the RNeasy column into a new 2 ml capture tube.

29.Add another 500 µl RPE buffer to the RNeasy column. Close the tube gently and centrifuge for 15 sec at full speed.

30.Place the RNeasy column in a new 2 ml capture tube and centrifuge in a microcentrifuge at full speed for 1 minute to “dry” the silica membrane.

31. Toml microcentrifuge tube.

32. Pipetthe RNeasy silica-gel membrane. DO NOT TOUCH THE SILICA GEL MEMBRANE. Look closely as you perform this step. Use both hands when pipeting, one on top and one down by the tip to guide the pipet. Make sure the water was evenly distributed on the membrane.

33. After allowing the water to incubate on the membrane at room temp for 1 minute

34. C35. Carefully remove the 30 μl which is recovered in the 1.7 ml

tube and pipet it back onto the center of the silica membrane of the same column.

36. Place column back in to the same 1.7 ml tube and spin again for 1 minute at full speed. This double elution ensures that the entire membrane was extracted.

37. Transfer the recovered RNA to a new 1.7 ml tube and label it with date, sample name, and what it is. Write clearly!!

38. Transfer 4 μl of RNA to a new tube for transport back to UVM for Nanodrop quantification and RNA assessment. Label this tube carefully with date, name, and RNA.

39. Keep the samples on ice and quantify samples using the spectrophotometer at a 1 to 50 dilution (1μl sample + 49μl H2O). This will be demonstrated by the instructor.

[Sample concentration:_________ ] [Write this on your tube]

40. Evaluatedone as a class. Pre-run the gel for 2 minutes and turn off. Add 14 μl of water to each well on the E -gel. Add 1μl of each sample to each well and pipet up and down to mix. Run the gel for 20 minutes.**Note: If no E-gel then proceede with section 5.2**

41.Samples should be labeled properly for return to UVM and analyzed using the Agilent Bioanalyzer 2100. This instrument is

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used to determine the condition of the RNA. Only fully intact RNA is eligible for microarray analysis. Why?

5.2 Quantifying & Evaluating Samples

1. Thaw your RNA samples on ice.2. Quantify samples using the spectrophotometer at a 1 to 50

dilution (1L sample + 49L sterile dH2O). Read at nm. Sample concentrations: Control: ___________ Treated: ___________

3. Prepare a 1.2% agarose gel. 4. Mix 200L of sterile dH2O with 3L of 6X loading dye and mix.5. Add 1L of your sample to the water/dye mix. 6. Load 20L to each well. 7. Load 20L of the 0.24-9.5Kb RNA ladder.8. Run the gel for 60 minutes at 100 Volts.9. Store your remaining samples at -20C.10. Stain your gel with Ethidium Bromide, visualize with UV

light, photograph and record results.Note: Ethidium Bromide is a carcinogen and mutagen. Wear

gloves!11. Your samples will be analyzed using the Agilent

Bioanalyzer 2100 to determine the condition of the RNA. Only fully intact RNA is eligible for Microarray analysis. See Appedix for sample bioanalyzer data.

5.3 Microarray Target Preparation; 1 st Strand cDNA Synthesis (DAY 2) Synthesis of double-stranded cDNA from total RNA is performed by priming off the poly-A tail of mRNA using T7 Oligo (d)T24. It is important to note that the 5s, 5.8s, 18s and 28s rRNA do not have a poly-A tail and will not be synthesized in this procedure. The sequence of T7 Oligo (d)T24 is:

5’ – [GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(d)T24] – 3’

Note: Spin all reagent tubes before starting as some have only 1 μl in them!!!!

1. Adjust 1-3 μg (determined by instructor) of RNA to 11μl with DEPC water in a RNase-free 0.5ml tube. Keep the tube on ice.

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2. Primer Hybridization: Combine the following reagents: a. T7 oligo d(T)24 2μlb. RNA(1-3 μg) 10μl

3. Vortex, and spin in microcentrifuge [full speed] for 5 seconds.4. Put in a thermocycler at 70°C for 10 minutes.

5. While the 70°C incubation is in progress prepare the following master mix. Add the following reagents IN ORDER to make the first strand master mix.

a. First Strand Buffer 5X 4 μlb. 0.1M DTT 2 μlc. 10mM dNTP 1 μld. Superscript II 1 μl

6. Vortex and spin in microcentrifuge [full speed] for 5 second and place on ice.

7. After the 70°C step, add the master mix to the RNA primer tube and incubate in a thermocycler at 42°C for 60 minutes.

8. After the incubation, place on ice. During this incubation, prepare the second strand master mix.

Second Strand cDNA Synthesis 9. Make the following master mix in a separate tube. Keep it on ice.

All tubes may need to be centrifuged to get the contents to the bottom of the tube because some tubes only contain 2ul of enzyme!

a. DEPC Water 91 μlb. 5x Second Strand Buffer 30 μl dNTP(10mM)

c. 3 μl E.coli DNA Ligase (10U/μl) 1 μl d. E.coli DNA Polymerase I (10U/μl) 4 μle. E.col i RNas e H (2U/μl ) 1

μl Total volume to be added to First Strand tube 130 μl

10. Vortex master mix and perform a 5 second quick spin in the microcentrifuge [full speed.

11. Add this 130 μl second strand master mix to first strand tube after the 42C incubation.

12. Vortex and perform a 5 second quick spin.

13. Incubate for 2 hours at 16°C in thermal cycler.

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14. At the end of 2 hours and while the sample is still at 16°C, add 2μl of T4 DNA polymerase and incubate for exactly 5 minutes. Mix well. Do not incubate longer then 5 minutes or the quality of the cDNA may decrease due to the 3’ to 5’ exonuclease activity of the polymerase.

15. At the end of the 5 minute incubation, add 10 μl of 0.5 M EDTA to stop the reaction.

16. Store sample at -20°C.Primer Hybridization: Combine 1L of T7 oligo dT and 11L of RNA (3g) in a 1.5mL microcentrifuge tube. Vortex and briefly spin at 4000rpm. Place in a thermocycler set at 70C for 10 minutes. Place on ice immediately after.

17. Combine 1L of T7 oligo dT and 11L of RNA (3g) in a 1.5mL microcentrifuge tube.

18. Vortex and briefly spin at 4000rpm. 19. Place in a thermocycler set at 70C for 10 minutes. Place

on ice immediately after.20. Add the following reagents to a 1.5mL microcentrifuge

tube:a. 1st Strand Buffer 5X 4L b. 0.1M DTT 2Lc. 10mM dNTP 1L

Vortex and briefly spin at 4000rpm. Place in a thermocycler set at 42C for 2 minutes.

21. Add 1L of Reverse Transcriptase (Superscript II).22. Vortex and briefly spin at 4000rpm. 23. Place in a thermocycler set at 42C for 60 minutes.24. After the incubation, appropriately label your tubes and

store at -20C.

5.4 Precipitating the cDNA, Cleaning the cDNA, and Setting Up the In Vitro Transcription (IVT) (DAY 3)

Note: Phenol Chloroform Isoamyl Alcohol is at a ratio of 25:24:1 and saturated with 10mM Tris-HCl pH 8. Phenol is an organic acid and care must be used when handling. Phenol can burn skin, be absorbed into the circulatory system and cause health problems. Chloroform is an organic solvent and should be used in the hood.

Phenol

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– Corrosive – Local anesthetic – Can severely damage eyes and skin– Can cause severe burns of the skin and eye– Poisonous if ingested-can be fatal

Chloroform– Can cause irreversible corneal injury– Fatal if swallowed– Flammable

Isoamyl Alcohol– Flammable– Poisonous

Precipitatin g t he cDN A: 1. Centrifuge a Phase Lock Gel tube at full speed for one minute.

DO NOT VORTEX.2. Add 162 μl of the bottom layer from the Phenol Chloroform

Isoamyl Alcohol (PCI) to the cDNA synthesis reaction and vortex for 2 seconds. Hold cap tightly as leaking can occur during the vortex.

Note: PCI tends to leak out of the pipet so work quickly.3. Transfer the cDNA-PCI mixture to the phase lock gel tube.

DO NOT VORTEX the phase lock gel tube.4. Centrifuge at full speed for 2 minutes.5. Transfer the top layer to a 1.7 ml microcentrifuge tube. 6. Add the following to the 1.7 ml microcentrifuge tube and vortex.

Ethanol (100%) 405 μlNH4OAc (7.5M) 80 μl Pellet Paint 1 μl

7. Centrifuge at 12000 x g [full speed] for 20 minutes at room temp.

8. GENTLY remove the tube from the Centrifuge being careful not to disturb the cDNA pellet. The pellet should be pink and approximately the size of a grain of salt. Put on ice and immediately proceed to next step. Keep your tube on ice.**At this point a tiny pink visible pellet should be present on the bottom of the tube. If not, see the instructor.**

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Cleaning the cDNA Pellet:9. Using a micropipet, carefully remove the liquid from the tube

being very careful not to disturb the pellet. Tip the tube to enable removal of as much liquid as possible. Remember that the pellet is your sample!

10. Add 500μl of ice cold [-20C] 80% ethanol to the tube with the pellet. Gently cap tube and invert slowly several times. Watch your pellet very closely. Place tube back in rack and let the pellet settle to the bottom of the tube for a minute. Alternately, you may Centrifuge the tube at full speed for 15 seconds to get the pellet back down to the bottom of the tube. [See video on website for this technique]

11. Using a 1 ml micropipet, carefully remove the ethanol being very careful not to disturb the pellet. Tip the tube to enable removal of as much liquid as possible.

12. Repeat steps 2 and 3 with a new aliquot of 80% ethanol.13. Finally, remove all of the ethanol. Centrifuge the tube

again [full speed] for about 5 seconds and using a smaller pipet such as a P20 or P200, remove the last few microliters. The goal is to remove as much ethanol as possible without disturbing you pelleted cDNA.

14. Place the tube with the pellet in a drying box for 10 minutes to evaporate all of the ethanol. The dried pellet is easily lost once it is dry. Be very careful to handle the tube gently. Close the cap gently. Visualize the dried pellet to confirm it is present in the tube.

15. Resuspend the pellet in 22 μl of DEPC water and put on ice.

I n Vi t ro Transcription (IVT): Synthesis of b iotin labeled cRNA from cDN A using the Enzo Ki t:

16. Using the ENZO bioarray kit, a master mix for the entire class will be prepared as follows. The instructor will prepare

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this mix or designate someone from the class. This must be done an RNase-free area free from air currents.

Amt/sampl e # Samples Total a. Reagent 1 [10x Reaction buffer] 4 μl b. Reagent 2 [10x Biotin nucleotides] 4 μl c. Reagent 3 [10x DTT] 4 μl d. Reagent 4 [10x RNase Inhibitor] 4 μle. Reagent 5 [20x T7 RNA polymeras e ] 2 μl

Total Volume 18 μl NOTE: Be sure to make extra for this step. Add enoμgh for 1 more reaction

17. Combine the following in a 0.5 ml microcentrifuge tube and pipet up and down several times to mix. Spin in Centrifuge full speed for 5 seconds.

a. Clean cDNA 22 μl b. Enzo master mix [from above ] 18 μl

Total Volume 40 μl18. Incubate the above mixture at 37°C for 16 hours in the

thermocycler. 19. Store the sample at -20 C after the 16 hour incubation is

complete (to be done by instructor).

5.5 Cleaning the Biotinylated cRNA Using the Qiagen RNeasy Kit and Fragmentation (DAY 4) Note: Add 100L BME per 1mL RLT Buffer. RLT Buffer is stable for 1 month after addition of BME. We shall call this reagent b-RLT. All steps of the RNeasy protocol should be performed at room temperature.

1. Transfer the cRNA sample to a 1.7 ml microcentrifuge tube and add 60 μl of DEPC water and 350 µl BME-RLT and vortex.

2. Add 250 µl ethanol (96–100%) and lightly vortex again.3. Apply the sample (700 µl) to an RNeasy mini column placed in a

2 ml collection tube (supplied). 4. Close the tube gently and Centrifuge for 15 s at full speed.5. Remove the pass-through liquid from the tube and reapply it to

the same RNeasy mini column again. 6. Close the tube gently and Centrifuge for 15 s full speed. Transfer

the RNeasy column into a new 2 ml collection tube and discard the flow-through and collection tube.

7. Pipet 500 µl of RPE buffer onto the RNeasy column. Close the tube gently, and Centrifuge for 15 s at full speed to wash the column.

8. Transfer the RNeasy column into a new 2 ml collection tube.

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Discard the flow-through and collection tube.9. Add another 500 µl RPE buffer to the RNeasy column. Close

the tube gently and Centrifuge for 15 s at full speed to dry the RNeasy silica-gel membrane.

10. TransferCentrifuge in a microcentrifuge at full speed for 1 min.

11. Transfer the RNeasy column intomicrocentrifuge tube.

12. To recover the RNA from the membrane, pipet 30 µl of DEPC water onto the RNeasy silica-gel membrane and wait 1 minute. Make sure the DEPC water contacts the entire membrane. Do not touch the membrane. Close the tube and Centrifuge for 1 min at full speed to elute.

13. Remove the eluted RNA and apply it to the membrane again. Close the tube gently and Centrifuge for 1 min at full speed to elute. This double elution results in a slightly higher cRNA yield.

14. Transfer the cRNA to a new microcentrifuge tube. 15. Quantify

a. Add 49 μl of water to a Uvette and add exactly 2 μl of sample.

b. Pipet up and down to ensure all cRNA has been expelled (2μl sample + 48 μl H2O).

c. Use a p200 pipet set to 25ul and mix by pipetting up and down to mix well. Tap out all bubbles from Uvette.

d. [Sample concentration:_________ ]

Calculating th e Adjuste d cRNA Concentration Calculation of the adjusted cRNA concentration must be done in order to subtract out the original untranscribed RNA [i.e. tRNA, 5s, 18s, and 25s rRNA]. The cRNA is fragmented using a metal-induced hydrolysis reaction to create cRNA fragments of 35-250 bp to be used in hybridization to the yeast GeneChip. Both the unfragmented and fragmented cRNA are visualized on an agarose gel. This is important because we need to ensure that the unfragmented cRNA is not degraded from contamination during the handling steps leading up to this point.

16. Calculate the Adjusted cRNA concentration using the following formμla:

a. ADJ cRNA= {Amount of clean cRNA} - {Total amt RNA used at start}

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b. Example: 52.9μg –2μg=50.9μg or 50,900ngc. A) What is the concentration of your cRNA in ug

[not ng] per μl: __________ ug/μld. B) How many μl do you have:__________μle. C) How many μg of total RNA did use on day

1:__________μgf. [AxB]-C = adjusted cRNA concentration

Fragmenting the cRNA for Target Pre paration Affymetrix recommends that the cRNA used in the fragmentation procedure be sufficiently concentrated to maintain a small volume during the procedure. This will minimize the amount of magnesium in the final hybridization cocktail. The cRNA must be at a minimum concentration of 600 ng/µL. The fragmentation buffer contains a basic solution of TRIS acetate, magnesium acetate, and potassium acetate which causes a metal induced RNA hydrolysis at high temperature.

17. Setup the following fragmentation reaction in a 0.5 ml Centrifuge tube. Use a 0.5 ml tube for this reaction

Adj cRNA concentration: __________ ng/μl 10μg=___________μl Volume Volume (μl)

10 μg of adj cRNA 1-16 μl5x fragmentation buffer 4 μl DEP C wate r ( as needed to make the final volume 20 μl) Total reaction volume 20 μl 20 μl

18. Vortex and Centrifuge briefly. 19. Incubate at 94°C for 30 minutes in a thermocycler.

Put on ice following the incubation.

Fragmentation: Hammerhead Cleavage

20.

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Fragmentation buffer 94C for 30 minutes

50-200 bp fragments

Lets look at the molecμlar level

21.22.

Evaluating The Samples on a Standard Agarose Ge l This procedure will evaluate your unfragmented and fragmented cRNA. Samples will be loaded on a precast E-GEL and allowed to run for 20 minutes.

23. Remove a E-Gel from pouch and place on E-Gel base. 24. Pre-run the gel for 2 minutes by pressing the button

continuously for 10 seconds. You will see the light start to blink indicating it is in a pre-run mode. It will beep and stop after 2 minutes.

25. Add26. Add

mix well. Run both the fragmented and unfragmented cRNA.27. Load 4 μl of Ladder28. Run the gel for 20 minutes.29. Visualize the E-gel on a transilluminator. 30. Take pictures.

Note: The fragmented sample should be a smeared band at the bottom of the gel indicating fragments of RNA between 35 and 250bp. The unfragmented sample should be a smear

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The intermediate product is a 2’, 3’ cyclic nucleotide. The next step is hydrolysis at the 3’ carbon.

The cyclic nucleotide is now a 2’-nucleotide. Hydrolysis at the 2’ carbon produces the 3’-nucleotide.

from the top to the bottom of the gel. The total RNA [if you run them] should be two bands at the middle of the gel indicating the 25s and 18s ribosomal RNA units. See picture below.

ladder 2000, 1200, 800, 400, 200, 100bp

The remainder of the experiment will be performed by the UVM DNA/Microarray Processing Facility. See p. 35 for a detailed summary of what procedures will be used to complete this experiment.

Day 5: Tour of Core FacilityDay 6: Tutorial of Bioinformatics with Dr. Janet Murray

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Appendix A: Solutions Guide

SOLUTIONS GUIDE0.5M NaOH/0.8M NaCl p. 730.5M Tris/ 1.5M NaCl p. 732.0M Tris Cl (pH 8.0) p. 69Amido Black Destain p. 78Amido Black Stain p. 78Ampicillin Stock p. 69Blocking Buffer p. 77Block. Buf. (0.1%(w/v) BSA) p. 79Buffer 1 p. 75Buffer 2 p. 75Buffer 3 p. 75CaCl2 (50 mM) p. 72Chelex Extraction Solution p. 80Chloroform:Isoamyl Alc. (24:1)p. 72Coomassie Blue Destain p. 77Coomassie Blue Stain p. 77Cracking Buffer p. 73Denhardt’s (100X) p. 74dNTPs mix p. 76Heat-treated RNase A p. 71Hybridization Solution p. 74LB amp p. 69Lysis Buffer p. 76PAGE Running Buffer p. 76Phenol/Chloroform/Isoamyl p. 80PBS (Phosphate Buffer) p. 71Potassium Acetate (5M) p. 71Prehybridization Solution p. 74

Primary Antibody Dilution p. 78

Primer Dilutions p. 75Resolving Buffer (4X) p. 76

SA-AP p. 75Sample Buffer (6X- for DNA) p. 70SDS Sample Buf. (6X -for pro.) p. 77Secondary Antibody p. 78SG Buffer p. 80Sodium Acetate (3M) p. 73Solution I p. 71Solution II p. 71SSC (20X) p. 70SSC 0.16X /0.1%(w/v) SDS p. 75SSC 0.2X /0.1% (w/v) SDS

p. 74SSC 2X /0.1% (w/v) SDS

p. 74Stacking Buffer (4X)

p. 76Sterile Saline (0.145M)

p. 72Sterile Saline (0.9%)

p. 79Stop Solution

p. 79TAE stock (50X) p. 69/70TBS (4X)

p. 70TBS-T

p. 70TE Buffer

p. 69

68

Transformation Buffer p. 72

Western Blot Transfer Buffer p. 77

YPD Nutrient Media p. 80

λHind III maker p. 70

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GENERAL SOLUTIONS

Ampicillin Stock100 mg/ml stock solution: for example, 0.5g ampicillin sodium salt into 5 ml dH20. Filter sterilize, and store at -20°C.

LB amp100 µg/ml final concentration: 1:1,000 dilution of ampicillin stock into LB broth. For example, add 1 ml ampicillian stock into 1 L LB broth.

*NOTE: Ampicillin is heat-sensitive, so LB broth must be cooled to 60°C after coming out of the autoclave, before the ampicillin is added. Setting the water bath to 60°C and letting the LB broth cool in there for an hour is a good way to ensure the LB doesn’t solidify (if making plates).

***1ul into 1ml for LB amp***

TE bufferNeed (final conc.):10 mM Tris-Cl (pH 7.5) 1mM EDTA (pH 8.0)Make: 1.576 g Tris-Cl

0.2922 g EDTABring up to 1 L dH20

2M Tris-Cl (pH 8.0)177.6g Tris-Cl10.6g Tris-baseIn ~950mL sterile dH20**Ph 8.0**Bring up to 1.0L with sterile dH20

50X TAE stock (pH 8.5)Prepare a Stock Solution of 0.5M EDTA:

93.05g EDTA disodium salt (FW= 372.2)In ~400ml sterile dH20***bring pH to 8.0***Up to 500mL with sterile dH20

Prepare a 50X Stock of TAE242g Tris Base (FW= 121.14)In ~750ml sterile dH20Carefully add 57.1mL Glacial Acetic Acid

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100mL 0.5M EDTA (pH 8.0)Bring up to 1L with sterile dH20

pH 8.5, but no adjustment needed*Dilute 50X stock 1:10 for a 5X stock*

6X DNA sample bufferNeed: 0.25% (w/v) Bromophenol Blue

40% (w/v) Sucrose Make: 0.25 g Bromophenol Blue

40 g Sucrose100 ml dH20

λ Hind III markerWant a final concentration of 100 ng/µL from Invitrogen stock.Make: 96 µL λ Hind III marker (Invitrogen stock)

320 µL TE68 µL 6X loading buffer

20X SSC (pH 7)175.3 g NaCl88.25 g Na3 Citrate•2H201 L dH20**pH 7.0**

4X TBS (pH 7.4)60.55 g Tris base4.0 g SDS1000 ml dH20

TBS-T1X TBS, 0.4% Tween-20

8 ml Tween-201992 ml 1X TBS

Phosphate Buffer (PBS)4.0g NaCl0.1g KCl0.72g Na2HPO4

0.12g KH2PO4

In ~400ml sterile dH20**pH 7.4**Bring to 500ml with sterile dH20

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MODULE 1

1.1: Isolation of plasmid DNA

Solution I0.50 g D-Glucose0.625 ml 2M Tris-Cl (pH 8)1 ml 0.5M EDTAAdd dH20 to make total volume 50ml**add 5 mg/ml lysozyme just before use**

Solution II2 ml 1M NaOH1 ml 10% SDS7 ml dH20**Prep fresh**

5M Potassium Acetate29.5 ml glacial Acetic Acid 100 ml dH20 Add KOH pellets until pH=4.8

Heat-treated RNase A (100mg/ml)Dissolve 100 mg (0.1g) of pancreatic RNase A in 1 ml 10mM Tris-Cl/15mM NaCl. Store at -20 ºC. Immediately before use, heat RNase A in 100°C heat block for 15 minutes and allow tubes to cool slowly to RT.

Salt Saturated PhenolTris buffered Phenol pH 6.6/7.98-Hydroxyquionoline added until dark yellow/orange color

Chloroform:Isoamyl Alcohol (24:1)480 ml Chloroform20 ml Isoamyl Alcohol

TE bufferSee general solutions section

1.4: Transformation

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50 mM CaCl20.73 g CaCl2

100 ml dH20** Autoclave to sterilize**

Transformation buffer1ml 100 mM CaCl2

1 ml 100 mM Tris1 ml 100 mM NaCl7 ml dH20

1.6: Secondary selection of transformed bacteria

0.145 M Sterile Saline (pH ~7)4.25 g NaCl500 ml dH20** Autoclave to sterilze**

1.8: Cracking gel

Cracking Buffer (pH 6.8) 0.788 g Tris-Cl1.0 g SDS0.058 g Na2EDTA•2H2013.6 g Sucrose0.1 g Bromophenol Blue100 ml dH20** pH 6.8**

1.9: Biotin labeling of DNA

3 M Sodium Acetate (pH 4.8)24.6 g Sodium Acetate100 ml dH20** pH 4.8**

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1.11: Southern blot

Cracking BufferSee 1.8 above

0.5M NaOH/0.8M NaCl20 g NaOH46.752 g NaCl1 L dH20

0.5M Tris/1.5M NaCl (pH 7)250 ml 2 M Tris-base solution87.6 g NaCl750 ml dH20** pH 7**

10X SSCSee general solutions for 20X SSC

1.13: Hybridization of Southern Blot

Prehybridization Solution (per group, prep fresh)5 ml Formamide2.5 ml 20X SSC0.5 ml 100X Denhardt’s solution (doesn’t keep more than 24

hours!)0.25 ml 1M Phosphate Buffer200 µL Herring sperm DNA, freshly denatured

Hybridization Solution (per group, prep fresh)4.5 ml Formamide 2.5 ml 20X SSC0.1 ml 100X Denhardt’s solution (doesn’t keep more than 24

hours!)0.4 ml 1M Phosphate Buffer1.5 ml dH2O200 µL Herring sperm DNA, freshly denaturedBiotin-labeled probe DNA

100X Denhardt’s Solution0.2 g Ficoll 0.2 g Polyvinylpyrrolidone0.2 g Bovine Serum Albumin (BSA)

74

10ml sterile dH20**Doesn’t keep more than 24 hours**

1.14: Detection of DNA

2X SSC/0.1% (w/v) SDS (per group)50 ml 20X SSC450 ml dH200.5 g SDS

0.2X SSC/0.1% (w/v) SDS (per group)5 ml 20X SSC495 ml dH200.5 g SDS

0.16X SSC/0.1% (w/v) SDS (per group)4 ml 20X SSC496 ml dH200.5 g SDS

1.15: Development of Blot

Buffer 1 : Final Concentration: 0.1 M Tris-Cl 0.15 M NaCl

8.7 g NaCl15.764 g Tris-Cl1 L dH20

Buffer 2: 3% (w/v) BSA in Buffer 13g BSA per 100 ml Buffer 1**Doesn’t keep more than 24 hours, prep fresh**

SA-AP: **Needs to be made immediately before use**1 µL SA-AP per 1 ml Buffer 2 (approx. 10 ml needed per group)

Buffer 3Final concentration: 0.1M Tris-Cl

0.1M NaCl 50 mM MgCl2

15.764 g Tris-Cl5.844 g NaCl10.15 g MgCl2

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1 L dH2O

1.17: PCR25 mM Primer Dilutions (from 500 mM stock primers)

25 µL 500mM stock475 µL dH20**Primers should be stored long term at 500 mM conc. and

diluted to 25 mM in smaller batches**

1.25 mM dNTP mix (from 100 mM individual dNTP stocks)600µL dCTP600µL dTTP600µL dATP600µL dGTP2.6 ml dH20

MODULE 2

2.2: SDS-PAGE prep and Pierce Protein Assay4X Resolving Buffer (pH 8.9)

18.17g Tris base10 g SDS100 ml dH20**pH 8.9**

Lysis Buffer5 ml 1M Tris (pH 8.0)3 ml 5M NaCl1 ml Triton 100X91 ml dH20**add 0.0057 g DTT/10 ml Lysis Buffer fresh immediately before

use**

2.3: SDS-PAGE4X Stacking Buffer (pH 6.8)

6.055 g Tris base0.4 g SDS100 ml dH20**pH to 6.8**

PAGE running buffer76

10.92 g Tris base51.36 g Glycine3.6 g SDS1800 ml dH20

6X SDS sample buffer15 ml Glyercol6.25 ml 2M Tris-Cl0.186Na2•EDTA2 g SDS0.1 g Bromophenol Blue78.75 ml dH20** 1% (v/v) β-Mercaptoethanol needs to be added fresh before

use**

Coomassie Blue Stain200 ml Methanol50 ml Glacial Acetic Acid1 g Coomassie Blue250 ml dH20

Coomassie Blue Destain200 ml Methanol 75 ml Glacial acetic Acid725 ml dH20

2.6: SDS-PAGE and Western Blot

Western Blot Transfer Buffer (pH 8.3)2.93 g Glycine5.81 g Tris base200 ml Methanol800 ml dH20**pH 8.3**

2.7: Blocking with primary antibody

Blocking Buffer50 g dry milk6.35 g Tris-Cl1.18 g Tris base8.77 g NaCl1L dH20

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TBS-TSee general solutions section.

Primary Antibody Dilution (1:5,000)0.1% Tween-20, 1% (w/v) dry milk

50 ml 1X TBS50 µL Tween-200.5 g dry milk10 µL Anti-GST antibody

2.8: Staining with Amido Black and detection using secondary antibody

Amido Black Stain112.5 ml Methanol5.0 ml Acetic Acid0.25 g Amido Black132.5 ml dH20

Amido Black Destain112.5 ml Methanol5.0 ml Acetic Acid132.5 ml dH20

Secondary Antibody Dilution (1:10,000)4 µL Secondary Antibody40 ml TBS-T

MODULE 3

3.2: Overlaying plates

TBS-TSee general solutions section.

3.3: Identifying Positive Colonies

Blocking Buffer (0.1% (w/v) BSA)50 g dry milk6.35 g Tris-Cl

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1.18 g Tris base8.77 g NaCl10 g BSA1000 ml dH20

Primary Antibody Dilution (1:5,000)0.1% Tween-20, 1% (w/v) dry milk

50 ml 1X TBS50 µL Tween-200.5 g dry milk10 µL Anti-GST antibody (produced in rabbit)

TBS-TSee general solutions section.

Secondary Antibody Dilution (1:10,000)4 µL Goat anti-rabbit IgG40 ml TBS-T

Stop Solution (pH 8.0)1.21 g Tris-Cl0.93 g EDTA500 ml dH20**pH 8.0**

MODULE 4

4.1: Making Template DNA and Performing PCR Reaction0.9% Sterile Saline Solution

9 g NaCl1000 ml dH20Autoclave to sterilize

10% Chelex Extraction Solution10 g Sigma Chelex 100 Resin100 ml dH20**prep fresh**

25 mM Primer Dilutions (from 500 mM stock primers)25 µL 500mM stock475 µL dH20**Primers should be stored long term at 500 mM conc. and

diluted to 25 mM in smaller batches**

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1.25 mM dNTP mix (from 100 mM individual dNTP stocks)600µL dCTP600µL dTTP600µL dATP600µL dGTP2.6 ml dH20

MODULE 5YPD Nutrient Media

50 g YPD Nutrient Media (Difco powder) 1L dH20**Autoclave to sterilize**

SG Buffer9.2 g Sorbitol1.8 g EDTA50 ml dH20**pH 7.4**

Phenol/Chloroform/Isoamyl Alcohol (25:24:1)25 ml Phenol24 ml Chloroform1 ml Isoamyl Alcohol**add 10mM Tris-Cl until pH=8**

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Appendix B: Sterile TechniqueIt is very important in microbiology and genetics to work with pure cultures. Unfortunately, this is difficult. The world around us is covered with microorganisms. Microorganisms are even carried on dust particles in the air. In order to protect sterile broth, plates, slants and pure cultures from the microbes all around us, we must practice sterile (aseptic) technique. This simple means that sterile surfaces or sterile media must be protected from contamination by microbes in the air or residing on non-sterile surfaces. A simple example of the problem is that a sterile petri plate can become contaminated with bacteria when the lid is removed. In sterile technique, only sterile surfaces touch other sterile surfaces and exposure to the air is kept to a minimum.

In the classroom, you often need to practice sterile technique when you inoculate a pure culture of a microorganism into fresh medium. Sometimes this is a transfer to a tube of liquid broth and at other times, it is a transfer to a petri plate-containing agar. While there are other circumstances that require sterile technique, these are the most common and they will be described in more detail on the pages that follow.

Appendix C: Spread Plate Technique

1. Dispense the appropriate volume of sample into the center of a sterile agar plate.

2. Dip the glass spreader (aka “hockey stick”) in alcohol. 3. Pass the spreader through the flame of a Bunsen burner to burn

off the alcohol. (This sterilizes the spreader).

***IMPORTANT***Keep the dish of alcohol behind the Bunsen burner.

Keep the alcohol dish covered when you are not using it.Keep your hand above the spreader at all times or flaming

alcohol may roll toward your hand.If the dish of alcohol catches on fire, cover the dish with the

glass lid and it will go out.

4. Cool the spreader by touching it to the agar where there is no sample.

5. Spread your sample over the entire surface of the agar.6. Sterilize the spreader before putting it back on the bench.

Appendix D: Use of a Rainin Pipettor

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Take note: Never rotate the volume adjustor beyond the upper or lower

range of the pipette man, as stated by the manufacturer. Never use the pipette man without the tip in place; this could

ruin the precision piston that measures the volume of fluid. Never lay down the pipette man with filled tip; fluid could run

back into the piston. Never let plunger snap back after withdrawing or ejecting fluid;

this could damage the piston. Never immerse the barrel of the pipette man in fluid. Never flame pipette man tips.

If you drop your pipette man, the precision piston system can be damaged; therefore, if your pipette man is dropped, be sure to check the pipetting accuracy has not been affected.

Recommended Volume Ranges:

Model p10: 0.5-10 μL, the number after the decimal point is in redModel p20: 1-20 μL, the number after the decimal point is in redModel p200: 20-200 μL, there is no decimal pointModel p1000: 200-1000 μL, the numbers after the decimal point are in black

Pipetting Directions – Method1. Set the desired volume by holding the pipette man body in one

hand and turning the volume adjuster knob until the correct volume shows on the digital indicator. Approach the desired volume by dialing downward from a larger setting.

2. Press tip onto shaft by a slight twisting motion.3. Depress the plunger to FIRST POSITIVE STOP. This part of the

stroke is the calibrated volume displayed on the digital micrometer.

4. Holding the pipette man vertically (never more than 20˚ from vertical), immerse the tip just below the level of the liquid.

5. Allow the pushbutton to return SLOWLY to the up position. Move the tip so that it stays slightly below the level of the liquid as you draw up.

6. Wait one to two seconds to ensure that the full volume of sample is drawn up into the tip.

7. Withdraw the tip from the sample liquid.8. To dispense the sample, place the tip end against the sidewall of

the receiving vessel and depress the plunger to the FIRST STOP. Wait one to two seconds. Then depress the plunger to the SECOND STOP, expelling any residual liquid in the tip.

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9. With the plunger fully depressed, withdraw the pipette man from the vessel carefully with the tip sliding along the wall of the vessel.

10. Let the plunger return slowly to the UP position. If an air bubble is observed, re-pipette the sample.

11. Pre-rinsing the tip with the liquid being pipetted is recommended. A significant film may be retained on the inside wall of the tip, resulting in an error. Since the film remains relatively constant in successive pipettings with the same tip, refilling the tip a second time and using this quantity as the sample may obtain good reproducible results.

12. Discard the tip by depressing the tip ejector button smartly in the appropriate waste container.

Appendix E: Pipette ExercisesPipette Exercise #1

Determine and record the pipettor best suited for each of the measurements listed below.Add the indicated amounts to labeled microfuge tubes. Use the matrix below as a checklist while adding solutions to each microfuge tube.

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Solution 1 Solution 2 Solution 3Tube A (green) 10 µL 25µL 0.0963 mlTube B (red) 2 µL 0.015 ml 183 µLTube C (blue) 598.6 µL 0.200 ml 201.4 µL

Determine the total volume being added to each of the tubes. To check that your measurements are accurate, set a pipettor to the final volume and carefully withdraw the solution from each tube. Is the tip just filled? If measurements are inaccurate, repeat the exercise to obtain a near-perfect result.

Pipette Exercise #2

Using each of the three pipettors that are available, perform the following:Set the pipettor to its maximum volume.Using water at room temperature, carefully pipette the water onto a weigh boat that you have tared (re-zeroed).Room temperature water has a density of approximately 1 gm/ml or 1 g/L. Therefore, you can determine the accuracy of your pipetting, e.g., 1000 L of water will weigh 1gm. Repeat the pipetting until you feel that you are reasonably accurate. Then record the weights of five successive pipettings. Determine the mean and standard deviation associated with your measurements.

Complete the following conversions:

1L =_______ml 0.001L =________ml10L =_______ml 0.11L =________ml100L =_______ml 0.01ml =________ml1000L =_______ml 1L =________ml

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APPENDIX F: PCR Reagents and Conditions

Cycling Program: GST New

94C 5 min Initial Elongation

94C 1 min5X50C 1 min

72C 1 min

94C 1 min25X51C 1 min

72C 1 min

72C 10 min Final elongation

4C HOLD ***************

SAMPLESInitial Stock Concen.

Components 1 2 3 4 5 6 7

 Template DNA* 1-10 1-10 1-10

3 coloni

es

3 coloni

es 1-10 1-10  10X Buffer 5 5 5 5 5 5 550mM MgCL2 3 6 9 3 3 3 31.25mM dNTPs 8 8 8 8 8 8 8

25uMForward Primer 1 1 1 1 1 XXX 1

25uMReverse Primer 1 1 1 1 1 1 XXX

5U/ulTaq Polymerase 0.5 0.5 0.5 0.5 0.5 0.5 0.5

  dH2O              

*NOTE: Samples 1-3, 6 & 7: Use PLASMID DNASample 4: Transformed ColoniesSample 5: Non-Transformed ColoniesDESIRED FINAL VOLUME: 50µL

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APPENDIX G: PIERCE PROTEIN ASSAYOD Value

L Alb (Stock 2mg/ml)

L dH2O ml Dye

[Alb g/ml] SeriesA

Series B

0 100 2 0 2.5 97.5 2 50 5.0 95.0 2 100 7.5 92.5 2 150 10.0 90.0 2 200 12.5 87.5 2 250 15.0 85.0 2 300

L Sample

L dH2O

ml Dye

OD Values g/ml in Cuvette

g/ml Original Solution

222222

APPENDIX H: Frequently Used DNA/Protein Markers

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Lambda DNA-Hind III Digest

APPENDIX I: GST Plasmid Map

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APPENDIX J: Streak Plate Method

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APPENDIX K: SDS-PAGE Assembly Instructions89

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APPENDIX L: Western Blot Assembly Instructions

91