Genetic Lab Notebook

08Fall

IndexRunning a Gel…………………………………………………….…………………..2

Isolation of genomic DNA and PCR.………………………………………..3

The Nucleosome Structure of Chromatin………………………………..6

Cloning satellite DNA………………………………………………………………8

Fall 2010

Genetics Lab Notebook

Genetics Lab NotebookKevin Chen

Lab 1: Running a Gel 9/24/2010

ObjectiveTo become familiar with the basic method of Agarose gel electrophoresis

Protocol Weight out 0.75g of Agarose and transfer to an Erlenmeyer flask. Measure out 75ml of 1xTAE buffer using graduate cylinder and add to solution to

Erlenmeyer flask. Boil the mixture solution to fully dissolve Agarose, then leave the solution to cool to

room temperature Add 8µL of ethidium bromide into the mixture solution and swirl to mix Pour the gel solution slowly into the tank. Push any bubbles away to the side using a

disposable tip. Insert the comb and double check that it is correctly positioned. Leave to set for at least 30 minutes with the lid on if possible until the gel solidifies. Pour 1xTAE buffer into the gel tank to submerge the gel to 2–5mm depth. This is the

running buffer. Load 8µL of each pre-prepared sample into each well. Load the first and second well with

molecular weight marker, 3rd and 4th well with EcoRI, 5th and 6th well with Hind III, and the 7th and 8th well with Pst1.

Close the gel tank, switch on the power-source and run the gel at 180v, check to ensure that a current is flowing

Monitor the progress of the gel by reference to the marker dye, when the process is completed in roughly 30 mins, Switch off and unplug the gel tank and carry the gel (in its holder if possible) to the dark-room to look at on the UV light-box.

Data and Discussion Lanes loaded with EcoRI showed bands at approximately 3.4kb, lanes loaded with PstI showed bands at 7.8kb, lanes loaded with HindIII showed bands at 5.6kb. The lighter protein would travel the less distance across the gel while the heavier ones travel further across the gel, therefore showing different bands at different locations on the gel.

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Lab 2: Isolation of genomic DNA and Chromosome PCR10/1/2010 & 10/8/2010

ObjectiveTo isolate genomic DNA and use PCR to specifically amplify PV92 locus on human chromosome 16 to determine genotype, as well as to compare DNA polymorphisms between individuals.

Protocol Pour prepared saline from the tube into the mouth, rinse vigorously for 30 seconds

before expelling the saline back into the tube. Transfer 1000 microliter or 1 ml of the oral rinse into a micro test tube, then spin the

tube in a balanced centrifuge for 2 minutes at full speed. Remove tube from centrifuge, pour off the supernatant on top and discard while taking

care not to lose any cell pellet at the bottom of the micro test tube. Note that if it okay for a small amount of saline to remain in the bottom of the tube

Resuspend the pellet thoroughly by flicking the tubes until no cell clumps remain. Transfer 20 ul of resuspended cells into a microtube containing InstaGene and scew the

caps tightly. Shake the tube several times to mix the contents and place the tube in a 56°C water bath

for 10 minutes. Be sure to take tube out and shake it several times at halfway point (5 minutes in).

Remove tube from the 56°C water bath, shake several times, and place the tube in a 100°C water bath for 5 minutes

Remove tube from the 100°C water bath and allow the tube to cool for 2 minutes. Vortex the tube for 10 seconds or tap the tube vigorously to mix.

Spin the tube in a centrifuge for 5 minutes to pellet the cell debris, leaving DNA in the supernatant.

Transfer 20ul of the supernatant to a clean PCR tube, make sure not to transfer any of your matrix beads into the PCR reaction as the reaction will be inhibited.

Add 20ul of the PCR master mix into the PCR tube, which is a mix of most of the ingredients or factors necessary for PCR to occur, and pipet to ensure mixture of contents.

Load sample into a PCR machine to allow the reactions to take place. Remove your PCR samples from the PCR machine and Make a gel using protocol from lab 1 Load 10ul of PCR samples into different wells: lane 1 and lane 10 are PCR samples, while

lane 6 (+/+) and lane 5 (-/-) are control Run the gel for approximately 30-40 minutes. Remove gel and examine the stained gels

for expected DNA bands.

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DataLane 6 = + controlLane 5 = - controlLane 1 & 10 = sample

Discussion

As seen from the gel picture, both sample DNA are homozygous, with the sample in lane one having insert on both copy of the allele and sample in lane ten having insert on neither copy of allele.

1. Why is it necessary to chelate the matal ions from solution during the boiling/lysis step at 100 degrees? What would happen if you did not use a chelating agent such as the InstaGene matrix? InstaGene matrix is made up of negatively charged microscopic beads that "chelate", or grab metal ions out of solution. It acts to trap metal ions, such as Mg2+, which are required as necessary cofactors in many enzymes, such as DNases. By chelating these cations, then, we are inactivating these enzymes. As a result, we are protecting our genomic DNA from degradation by cellular enzymes that have been released during the DNA isolation protocol. If chelating agent was not used, our genomic DNA would become degraded and the experiment would fail.

2. What is needed from the cells for PCR?Cell’s double stranded DNA as the template for PCR

3. What structure must be broken to release DNA from a cell?Cell’s nucleus where double stranded DNA resides must be broken in order to release DNA from a cell. Cell’s plasma membrane must also be broken to release DNA.

4. Why do you think DNA is stored with cold InstaGene matrix after boiling the samples? Because InstaGene matrix serves to prevent the degradation of genomic DNA by DNases released during genomic DNA isolation. Therefore after boiling the samples and deactivating enzymes released by cells itself, DNA is preserved and becomes stored with cold InstaGene matrix.

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5. Why is it necessary to have a primer on each side of the DNA segment to be amplified? Primer is necessary for the replication of DNA. Having a primer on each side of the DNA segment to be amplified would allow replication taking place in the region and therefore amplifying that region.

6. How did Taq DNA polymerase acquire its name? The Taq name is a shortened for Thermophilus aquaticus, a thermophilic bacteria that is the source of the particular DNA polymerase enzyme. The enzyme heat resistant property is desired because it could withstand the high temperature during the PCR process.

7. Why are there nucleotides (A,T,C,G) in the master matrix? What are the other component of the master matrix and what are their functions?dNTPs are required for the replication of DNA as they are the structural unit of each strand of DNA. The master matrix should also contain Taq DNA polymerase, two different primers, one for each strand with sequence complementary to the DNA sequence, buffer in order for PCR to correctly take place.

8. Describe the three main steps of each cycle of PCR amplification and what reactions occur at each temperature. Step 1: the double-stranded DNA template is denatured and separate from each otherStep 2: oligonucleotide primers are annealed to the single-stranded DNA (ssDNA) template. One primer is designed to anneal to a specific region on the left side of one of the strands of DNA and the other primer is designed to anneal to a specific region on the right side of the complementary strand of DNA, thus amplifying the desired region.Step 3: DNA polymerase Taq extends each primer in the 5' to 3' direction, duplicating the DNA fragment between the primers. With each cycle of denaturing, annealing, and synthesis the specific DNA fragment is amplified exponentially.

9. Explain why the precise length target DNA sequence doesn’t get amplified until the third cycle. In the first polymerization step, a new DNA strand is synthesized onto each primer. Taq polymerase extends this new strand well beyond the target sequence, producing a new DNA strand that is longer than the target. In the second cycle, the DNA is denatured and primers anneal to the original target DNA and to the new DNA strands produced in the first cycle. A mixture of small target sequences and large hybrid DNA sequences are produced at the end of the second cycle. However in the third cycle, most of the templates have been created from the first two cycles, and contain DNA that has one primer incorporated, but nothing upstream of that position. In effect, the polymerases that extended primers used in the 3+ cycle will "fall off" the DNA where the opposite primer once sat. By the time you reach the end of your PCR reaction, most of your resulting amplified DNA will be only the length between the two primers of interest.

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Lab 3: The Nucleosome structure of Chromatin10/22/2010 & 10/29/2010

ObjectiveTo isolate Chromatin into DNA and histones

Protocol

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Data

Discussion:When nuclei are incubated with micrococcal nuclease, the enzyme cleaves the linker DNA between nucleosomes (the string) but not the nucleosomal core DNA (the beads). When the DNA isolated from these nuclei is electrophoresed on agarose gels, the DNA exhibits a ladder pattern with the length of the DNA in each band representing a multiple of the nucleosomal unit. The ladder pattern can be clearly observed on the gel obtained.

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Lab 4: Cloning Satellite DNA

Week 1: restriction digestion 11/5/2010

ObjectiveTo perform restriction digest on sheep satellite DNA and run a gel to separate individual fragments.

Protocol Digestive sheep DNA using digestive enzyme EcoRI. For each 17 microliters of sheep

DNA, use 13 microliters of restriction enzyme Pour a 1% argose gel Load 15 ul of each sample in each well as follow

1-2. Marker 3-4: sample 1 5. Sample 2 8: Sample 3

Run the gel for 30-45 min Take a picture (look for bands 370/430/800 )

Data

Discussion In all samples, three unique bands can be observed (800bp, 430bp, and 330bp) as the result of restriction digestion by EcoRI. One of these bands will be cut out for cloning.

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Week 2: DNA purification and ligation11/12/2010

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Week 3: Bacteria Transformation and Screening12/10/2010

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DataAs shown, no colonies were observed on the X gal-ampicilin agar plate

DiscussionTheoretically, bacteria cells that have taken up plasmid during transformation should show colonies on the plate due to the antibiotic resistance provided by the gene in the plasmid. Bacteria cells with plasmids that have the sheep DNA insertion should also give white colonies because the Lac gene in the plasmid that is able to catalyze X-gal into blue pigment would be disrupted by the insertion, while bacteria cells with plasmids that have no insertion show give blue colonies since Lac gene is still intact and undisrupted.

However, on the plate, there are no colonies of any kind. This most likely due to the fact that bacteria cells have not taken up any plasmids during transformation and therefore does not have ability to grow on ampicilin plate due to the lack of antibiotic resistance. Another likely reason is that the bacteria cells are incompetent to grow therefore even transformation took place correctly there still would not be any colonies on the plate.

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Genetic Lab Notebook