Mini-prep Isolation of Plasmid DNA - Santa Monica Collegehomepage.smc.edu/bober_mary/Bio 22/Mini-Prep plasmid DNA 202.pdf · Mini-prep EDVO-Kit # Isolation of Plasmid DNA EVT 001171K

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®

All components are intended for educational research only. They are not to be used fordiagnostic or drug purposes, nor administered to or consumed by humans or animals.

202EDVO-Kit #Mini-prep

Isolation ofPlasmid DNA

EVT 001171K

Storage:Store entire experiment in refrigerator

Experiment Objective:

The objective of this experiment is to introduce theprinciples of extracting plasmid DNA from bacterial cells.

Students will develop an understanding of the structure and function of plasmid DNAs.

2

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. Thisdocument, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.Copyright © 1997, 1998, 2000, 2001, EDVOTEK, Inc., all rights reserved. EVT 001171K

EDVO-Kit # 202: Mini-prep Isolation of Plasmid DNA

EDVOTEK • The BiotechnologyEducation Company ®

PageExperiment Components 2Experiment Requirements 3Background Information 5Experimental Procedures 9Study Questions 20

Instructor's GuideGeneral Information 21Pre-Lab Preparations 22Electrophoresis Hints and Help 26Idealized Schematic of Results 29Answers to Study Questions 30Material Safety Data Sheets 31

Major Section Headings

This experiment contains reagents for 20 plasmid isolations (Mini-preps) and enough electrophoresis reagents to prepare and run fiveagarose gels based upon the use of Horizontal gel electrophoresisapparatus, Model #M12.

ContentsA Tris Buffer concentrateB Sodium Hydroxide solutionC SDS solution (Sodium dodecyl sulfate, 10%)D Potassium acetate neutralization bufferE RNase (DNAse-free)

• Plasmid Extraction LyphoCells™ (freeze-dried)• 10x Gel Loading Solution• Practice Gel Loading Solution• UltraSpec-Agarose™ powder (2.5 g.)• 50x concentrated electrophoresis buffer• DNA Blue InstaStain™ sheets• 10x concentrated Methylene Blue Plus™ stain• 1 ml pipets• 100 ml plastic graduated cylinder• Microcentrifuge tubes• Microtipped Transfer Pipets

Experiment Components

Storage:Store entire experiment

in the refrigerator.

None of the experimentcomponents have been prepared

from human sources.

UltraSpec-Agarose, LyphoCells,Methylene Blue Plus,

and DNA Blue InstaStainare trademarks of EDVOTEK, Inc.

3




Experiment Requirements

PLASMID ISOLATION

• Microcentrifuge• Water bath (37°C)• Automatic micropipets with tips• Pipet pumps• 95-100% isopropanol• Ice

AGAROSE GEL ELECTROPHORESIS

• Horizontal gel electrophoresis apparatus• D.C. power supply• Automatic micropipets with tips• Hot plate, Bunsen burner or microwave oven• Recommended equipment:

DNA visualization system (white light)Staining Tray and Net

• 5 or 10 ml pipets• Pipet pumps• 250 ml beakers or flasks• Hot gloves• Marking pens• Distilled or deionized water

4




Experiment at a Glance

OPTION 1

Procedures for Plasmid Isolation start on page 9

EXPERIMENT #202ISOLATION OF PLASMID DNA

OPTION 2Requires Cat. # 622

Deproteinization Matrix

Procedures for Plasmid Isolation start on page 11

RESTRICTION ENZYMEDIGESTION

Requires Restriction Enzymes,not included with experiment.

AGAROSE GELELECTROPHORESIS

Experimental ProceduresPage 13

AGAROSE GELELECTROPHORESIS

Experimental ProceduresPage 13

DNA STAINING,VISUALIZATION AND

ANALYSISPage 17

DNA STAINING,VISUALIZATION AND

ANALYSISPage 17

PROTOCOL

5



EDVOTEK • The BiotechnologyEducation Company ® BACKGROUND INFORMATION

Isolation of Plasmid DNA

Many types of bacteria contain plasmid DNA. Plasmids are extrachro-mosomal, double-stranded circular DNA molecules generally contain-ing 1,000 to 100,000 base pairs. Even the largest plasmids are consid-erably smaller than the chromosomal DNA of the bacterium, whichcan contain several million base pairs. Certain plasmids replicate in-

dependently of the chromoso-mal DNA and can be presentin hundreds of copies per cell.A wide variety of genes havebeen discovered in plasmids.Some of them code for antibi-otic resistance and restrictionenzymes. Plasmids are ex-tremely important tools in mo-lecular cloning because theyare useful in propagating for-eign genes. When plasmidsare used for these purposes,they are referred to as vectors.

Through the use of recombinant DNA technology, hundreds of artifi-cial vectors have been constructed from elements of naturally occur-ring plasmids. These vectors have specifically designed properties thatmake them useful in solving particular experimental problems. Forexample, synthetic oligodeoxynucleotide linkers have been incorpo-rated into many plasmid vectors. These linkers contain many differ-ent restriction enzyme recognition sites to facilitate the insertion of for-eign DNA. The link-ers are often placednear characteristicmarker genes or highefficiency transcrip-tional promoters,both of which aid inthe isolation and ex-pression of thecloned DNA.

Plasmid DNA natu-rally exists as a super-coiled molecule. Supercoiling arises from alterations in the winding ofthe two DNA strands around each other. In certain areas of the mol-ecule, the DNA strands are wound around each other less frequentlythan in non-supercoiled DNA. The strain caused by these alterations

SupercoiledDNA

RelaxedPlasmid

DNA

"Nicks" will convert supercoiled DNAto circular form.

6



EDVOTEK • The BiotechnologyEducation Company ® BACKGROUND INFORMATION

Isolation of Plasmid DNA,continued create deformations in the DNA. These deformations partially relieve

the strain and ultimately lead to supercoiling. Supercoiled DNA isfolded onto itself and has a more condensed and entangled structurethan the same DNA which is relaxed. As an analogy to supercoiling,consider a rubber band. When the rubber band is twisted, it eventu-ally becomes knotted and collapses onto itself as an entangled ball.

Purified DNA must be a covalently closed circle to exist as a super-coiled molecule. Supercoiled plasmid DNA is often called Form I DNA.Supercoiling in the cell is caused by the action of enzymes called DNAgyrases. These enzymes use the chemical energy in ATP to introducesupercoiling into a relaxedmolecule. In addition, thereare enzymes that relax super-coiled DNA and are called un-winding or relaxing enzymes.Supercoiling has importantbiological consequences. Verylarge DNA molecules wouldsimply not fit in the cell if theywere not supercoiled. Geneexpression can also be influ-enced by supercoiling.

If one or more phosphatebonds anywhere in the back-bone of supercoiled DNA arebroken, the molecule unravelsto a relaxed form called opencircular DNA or Form II DNA.These breaks in the phosphate backbone are called nicks. Nickeddouble-stranded DNA is not covalently closed.

The two strands of nicked DNA are still held together by hydrogenbonds between the bases. With time, purified supercoiled DNA slowlydevelops nicks and converts to Form II. This is because supercoiledDNA is not as stable as its relaxed or open circular forms. Endonu-cleases, such as DNAse I, will randomly nick supercoiled DNA whenused in low amounts. Nicking can also be introduced by mechanicalmanipulations during plasmid purification.

During replication, several of the same plasmid molecules can forminterlocked rings. These multimers of plasmid are called catenanes. Acatenane containing two of the same plasmid molecules is called adimer. Similarly, those containing three or four molecules are calledtrimers and tetramers, respectively. Each plasmid molecule in a cate-nane can be supercoiled, however, for clarity, they are represented asrelaxed circles.

Dimer

Trimer

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BACKGROUND INFORMATION

Agarose gel electrophoresis is a powerful separation method frequentlyused to analyze plasmid DNA. The agarose gel consists of microscopicpores that act as a molecular sieve. Samples of DNA can be loadedinto wells made in the gel during molding. When an electric field isapplied, the DNA molecules are separated by the pores in the gel ac-cording to their size and shape. Generally, smaller molecules passthrough the pores more easily than larger ones. Since DNA has a strongnegative charge at neutral pH, it will migrate towards the positive elec-trode in the electrophoresis apparatus. The rate at which a given DNAmolecule migrates through the gel depends not only on its size andshape, but also on the type of electrophoresis buffer, the gel concentra-tion and the applied voltage. Under the conditions that will be usedfor this experiment, the different forms of the same plasmid DNA mole-cule have the following rates of migration (in decreasing order):

Supercoiled > linear > Nicked Circles > dimer > trimer > etc.

Supercoiled DNA has the fastest migration rate of the different formsof plasmid. In the plasmid extraction experiment you will be doing,there will be some residual, degraded RNA which consists of transferRNA and digested ribosomal and messenger RNA. Degraded RNAhas a faster migration rate than supercoiled plasmid DNA because it ismuch smaller in size.

In the first step of the experiment a cell lysis solution is added to thecells. This solution contains the detergent sodium dodecyl sulfate (SDS)which dissolves the cell membrane and denatures proteins. The solu-tion is very alkaline (pH > 12) due to the presence of sodium hydrox-ide. The high pH aids in denaturing proteins and causes the cleavageof the phosphate bonds in RNA. This eliminates interference from highmolecular weight RNA during the plasmid purification. Under highlyalkaline conditions, the two strands in non-supercoiled DNA (linearfragments of chromosomal DNA, relaxed and nicked circular DNA)separate and are partially removed from solution. However, this doesnot occur with supercoiled forms of plasmid DNA because the twostrands are intertwined and entangled in a way that prevents themfrom coming apart. Therefore, supercoiled plasmid remains free insolution.

The potassium acetate neutralization buffer contains acetic acid andpotassium salts. The acidic buffer neutralizes the alkaline conditionscreated by the sodium hydroxide. The potassium causes the SDS, withits associated membrane fragments and proteins, to precipitate. Thechromosomal DNA of E. coli is attached at several points to the cellmembrane. Centrifugation of the potassium-SDS-membrane complexesalso removes large amounts of entrapped chromosomal DNA.

Isolation of Plasmid DNA,continued

8




The addition of isopropanol precipitates the plasmid and remainingRNA. Tris buffer (diluted buffer concentrate for RNase) is used to re-suspend the DNA precipitate in a higher concentration. The buffercontains the enzyme RNase, which further degrades RNA. The con-centrated gel loading solution prepares the sample for electrophoresisby making it denser than the electrophoresis buffer. This enables thesample to sink into the wells of the submerged gel. A negativelycharged, blue tracking dye is also present to monitor the electrophore-sis and to make sample loading easier.

In this experiment, a 3000 base pair plasmid will be extracted from E.coli cells. The restriction map for this plasmid has a single site for EcoRI. Digestion with the enzyme will yield a single band measuring 3,000± 300 nucleotides. The multiple forms of the plasmid will be convertedto the linear form.

Isolation of Plasmid DNA,continued

BACKGROUND INFORMATION

9



EDVOTEK • The BiotechnologyEducation Company ® EXPERIMENTAL PROCEDURES

Option 1 - Isolation of Plasmid DNA for Detection on Gels

EXPERIMENT OBJECTIVE:

The objective of this experiment is to introduce the principles ofextracting plasmid DNA from bacterial cells. Students will developan understanding of the structure and function of plasmid DNAs.

LABORATORY SAFETY

This experiment is designed for staining of DNA with either DNA BlueInstaStain™ or Methylene Blue Plus™ stain after electrophoresis. Aswith any biological stain, care should be taken when handling solu-tions or gels containing methylene blue. Gloves and goggles shouldbe worn when handling staining reagents, and worn routinely through-out the experiment as good laboratory practice.

ISOLATION OF PLASMID DNA FOR DETECTIONON GELS

1. Obtain a microcentrifuge tube of suspended E. coli cells and putyour initials or group number on it. Place the tube on ice.

2. Using a designated 1 ml pipet, add 0.4 ml of Cell Lysis Solution(contains sodium hydroxide and SDS). This step will denature theproteins.

3. Cap the tube and gently mix by inverting the tube 5 times.

4. Keep on ice for 5 minutes.

5. Using a designated 1 ml pipet, add 0.3 ml of potassium acetateneutralization buffer (D). Potassium salt of SDS will precipitatefrom solution in the cold. This step will remove the SDS.

6. Cap the tube and mix thoroughly by inverting the tube. A whiteprecipitate should form.

7. Keep the tube on ice for 4 minutes.

8. Place the tube in a microcentrifuge with a counterbalance. (An-other group’s tube will serve this purpose, or a tube containing0.7ml of water .)

9. Centrifuge at full speed (10,000 to 14,000 rpm) for 5 minutes.

Place the 95-100%Ethanol orIsopropanol on icebefore the lab starts.

Allow adequate time to equilibratea water bath at 37°C for step 21.

Use a fresh pipet when going intodifferent stock solutions to avoidcross contamination.

Important Note:

Plasmid preparations from thisprocedure will not be effectivelydigested with restriction enzymes.Additional deproteinization isnecessary, and can beaccomplished using EDVOTEKCat. # 622, Deproteinization Matrix.Follow the Module 2 experimentalprocedures.

Wear Safety Goggles & Gloves

Remember!

10




Isolation of Plasmid DNA forDetection on Gels,continued

Step 10:After centrifugation,the precipitate willappear as a fluffymaterial adhering to the wall ofthe microcentrifuge tube.

Try to avoid touching the insidewall of the tube when transferringthe supernatant into a freshmicrocentrifuge tube.

Useful Hint!

Step 16:Be careful not to dislodgethe pellet and aspirate itinto the transfer pipet.

Remember!

10. When centrifugation is finished, pipet 0.5 ml of the supernatant(which contains plasmid DNA) into a fresh microcentrifuge tube.

Try to avoid transferring any of the precipitate, which will appearas a fluffy material adhering to the wall of the microcentrifuge tube.

11. Label the tube with your initials or group number.

12. Using a designated pipet, add 1 ml of ice cold 95-100% ethanol orisopropanol. Mix thoroughly by inverting and shaking.

13. Place the tube on ice for 5 minutes.

14. Place the tube (with counterbalance) in the microcentrifuge so thestrap which connects the lid to the tube faces the outside of therotor.

15. Centrifuge at full speed for 10 minutes.

After centrifugation, a small pellet should be visible in the lowerpart of the tube.

16. Carefully remove the supernatant with a transfer pipet withouttouching the inside walls of the tube.

17. Leave the tube open and allow the pellet to air dry for approxi-mately 10 minutes.

18. Resuspend the nucleic acid pellet by adding 50 µl of RNase Solu-tion (Tris-HCl-EDTA buffer containing RNase).

19. Cap the tube and mix by inverting, shaking or vortexing.

20. Briefly centrifuge to get all of the contents to the bottom of thetube.

21. Incubate the tube at 37°C for 10 minutes for the RNase reaction todigest RNAs that would interfere with the gel separation. Plas-mid DNA is not affected by RNase since the enzyme is specific forRNA only.

23. Prepare the sample for electrophoresis:• Add 5 µl of 10x Gel Loading Solution and mix.• Load 40 µl of the prepared sample on a 0.8% UltraSpec-

Agarose gel.

OPTIONAL STOPPING POINT

Store samples at 4°C until ready for electrophoresis.

11




Option 2 - Isolation of Plasmid DNA for Restriction Enzyme Digestion

Place the 95-100%Ethanol orIsopropanol on icebefore the lab starts.

Allow adequate time to equilibratea water bath at 37°C for step 24.

Use a fresh pipet when going intodifferent stock solutions to avoidcross contamination.

EXPERIMENT OBJECTIVE:

In this experiment, you will extract a 3000 base pair plasmid from E. colicells. The plasmid contains a gene for ampicillin resistance and ispresent in many copies per cell. Using the procedure outlined below,this plasmid can be digested with restriction enzymes.

Note: The extracted plasmid does not contain sites for common restrictionenzymes suitable for mapping purposes. EDVOTEK recommends Cat. #s206 or 306 which are experiments specifically designed for mapping.

LABORATORY SAFETY

This experiment is designed for staining of DNA with either DNA BlueInstaStain™ or Methylene Blue Plus™ stain after electrophoresis. Aswith any biological stain, care should be taken when handling solu-tions or gels containing methylene blue. Gloves and goggles shouldbe worn when handling staining reagents, and worn routinely through-out the experiment as good laboratory practice.

ISOLATION OF PLASMID DNA FOR RESTRICTIONENZYME DIGESTION WITH ECO RI

1. Obtain a microcentrifuge tube of suspended E. coli cells and putyour initials or group number on it. Place the tube on ice.

2. Using a designated 1 ml pipet, add 0.4 ml of Cell Lysis Solution(contains sodium hydroxide and SDS). This step will denature theproteins.

3. Cap the tube and gently mix by inverting the tube 5 times.

4. Keep on ice for 5 minutes.

5. Using a designated 1 ml pipet, add 0.3 ml of potassium acetateneutralization buffer (D). Potassium salt of SDS will precipitatefrom solution in the cold. This step will remove the SDS.

6. Cap the tube and mix thoroughly by inverting the tube. A whiteprecipitate should form.

7. Keep the tube on ice for 4 minutes.

Important Note:

These Experimental Proceduresrequire Deproteinization Matrix inorder to isolate plasmids suitablefor digestion with restrictionenzymes. DeproteinizationMatrix, EDVOTEK Cat. # 622needs to be purchasedseparately.


Remember!

12




8. Place the tube in a microcentrifuge with a counterbalance.(Another group’s tube will serve this purpose, or a tube contain-ing 0.7 ml of water.)

9. Centrifuge at full speed (10,000 to 14,000 rpm) for 5 minutes.

10. Transfer 0.5 ml of the supernatant to a clean microcentrifuge tube.The supernatant contains the plasmid to be further purified.

Try to avoid transferring any of the precipitate, which will appearas a fluffy material adhering to the wall of the microcentrifuge tube.

11. Mix the Deproteinization Matrix thoroughly and add 0.5 ml to thetube containing 0.5 ml of plasmid DNA solution.

12. Mix vigorously (or vortex) for 3 minutes.

13. Centrifuge at maximum speed for 5 minutes.

14. Transfer 0.5 ml supernatant to a clean microcentrifuge tube andadd 1 ml of 95-100% Ethanol or Isopropanol. Mix by inverting andshaking.

15. Place on ice for 5 minutes.

16. Centrifuge at maximum speed for 10 minutes.

17. Carefully remove the supernatant with a transfer pipet withouttouching the inside walls of the tube.

Rinse pellet with 1 ml of ice cold 80% ethanol to remove salts. (Becareful - pellet may dislodge.) Remove ethanol with a transfer pi-pet.

18. Leave the tube open and allow the pellet to air dry for approxi-mately 10 minutes.

19. Resuspend the nucleic acid pellet by adding 50 µl of RNase Solu-tion (Tris-HCl-EDTA buffer containing RNase, 0.08 mg/ml).

20. Cap the tube and mix by inverting, shaking or vortexing.

21. Briefly centrifuge to get all contents to the bottom of the tube.

22. Incubate the tube at 37°C for 20 minutes for the RNase reaction todigest RNAs that would interfere with the gel separation. Plas-mid DNA is not affected since the enzyme is specific for RNA only.

23. Freeze sample(s) to store, or proceed with restriction enzyme analy-sis. Assume plasmid DNA concentration to be approximately0.02µg/µl or 1µg per tube of isolated plasmid DNA.

Isolation of Plasmid DNA forrestriction enzymedigestion, continued

Step 10:After centrifugation,the precipitate willappear as a fluffymaterial adhering to the wall ofthe microcentrifuge tube.

Try to avoid touching the insidewall of the tube when transferringthe supernatant into a freshmicrocentrifuge tube.

Useful Hint!

Remember!

Step 17:Be careful not todislodge the pelletand aspirate it intothe transfer pipet.

13




Agarose Gel Electrophoresis


PREPARING THE GEL BED

Using 7 x 7 cm Gel Beds

1. Close off the open ends of a clean and drygel bed (casting tray) by using rubber damsor tape.

A. Using Rubber dams:• Place a rubber dam on each end of the bed. Make sure the

rubber dam sits firmly in contact with the sides and bot-tom of the bed.

B. Taping with labeling or masking tape:• With 3/4 inch wide tape, extend the tape over the sides

and bottom edge of the bed.• Fold the extended edges of the tape back onto the sides

and bottom. Press contact points firmly to form a goodseal.

2. Place a well-former template (comb) in thefirst set of notches nearest the end of the gelbed. Make sure the comb sits firmly andevenly across the bed.

Using the 7 x 15 cm Gel Bed for Two Gels

1. Close off the open ends of a clean and dry gel bed (casting tray) byusing rubber dams or tape.

A. Using Rubber dams:• Place a rubber dam on each end of the bed. Make sure the

rubber dam sits firmly in contact with the sides and bot-tom of the bed.

B. Taping with labeling or masking tape:• With 3/4 inch wide tape, extend the tape over the sides

and bottom edge of the bed.• Fold the extended edges of the tape back onto the sides

and bottom. Press contact points firmly to form a goodseal.

2. Place a well-former template (comb) in the first set of notches near-est the end of the gel bed. Place a second comb in the middle set ofnotches. Make sure combs sit firmly and evenly across the bed.

14




Casting Individual 0.8% Gels,continued

3. Use a 250 ml flask to prepare the diluted gel buffer.

• With a 1 ml pipet, measure the buffer concentrate and add thedistilled water as indicated in Table A.

4. Add the required amount of agarose powder. Swirl to disperseclumps.

5. With a marking pen, indicate the level of the solution volume onthe outside of the flask.

6. Heat the mixture to dissolve the agarose powder. The final solu-tion should be clear (like water) without any undissolved particles.

A. Microwave method:• Cover flask with plastic wrap to minimize evaporation.• Heat the mixture on High for 1 minute.• Swirl the mixture and heat on High in bursts of 25 sec-

onds until all the agarose is completely dissolved.

B. Hot plate or burner method:• Cover the flask with foil to prevent excess evaporation.• Heat the mixture to boiling over a burner with occasional

swirling. Boil until all the agarose is completely dissolved.

7. Cool the agarose solution to 55°C with careful swirl-ing to promote even dissipation of heat. If detectableevaporation has occurred, add distilled water tobring the solution up to the original volume asmarked on the flask in step 5.

Size of EDVOTEKCasting Tray

7 x 7 cm

7 x 15 cm

10.5 x 14 cm

Amt of Concentrated Distilled TotalAgarose + Buffer (50x) + Water = Volume

0.24 gm 0.6 ml 29.4 ml 30 ml

0.48 gm 1.2 ml 58.8 ml 60 ml

0.8 gm 2.0 ml 98.0 ml 100 ml

Individual 0.8 % UltraSpec-Agarose™ Gels for staining with DNA Blue InstaStain™Table A:


CASTING THE GEL

This experiment requires a 0.8% gel.

55˚C

Useful Hint!

At high altitudes, it isrecommended to usea microwave oven toreach boiling temperatures.

15




Casting Individual 0.8% Gels,continued

EXPERIMENTAL PROCEDURES

DO NOT POUR BOILING HOTAGAROSE INTO THE GEL BED.

Hot agarose solution may irreversibly warp the bed.

Caution!

After the gel is cooled to 55°C:

If using rubber dams, go to step 9. If using tape, continue with step 8.

8. Seal the interface of the gel bed and tape to prevent the agarosesolution from leaking.• Use a transfer pipet to deposit a small amount of cooled

agarose to both inside ends of the bed.• Wait approximately

1 minute for the agarose to solidify.

9. Pour the cooled agarose solution into thebed. Make sure the bed is on a level sur-face.

10. Allow the gel to completely solidify. It will become firm and coolto the touch after approximately 20 minutes.

Step 11: Be carefulnot to damage or tearthe gel when removingrubber dams. A thin plastic knifeor spatula can be insertedbetween the gel and the dams tobreak possible surface tension.

Useful Hint!

PREPARING THE GEL FOR ELECTROPHORESIS

11. After the gel is completely solidified, carefully and slowly removethe rubber dams or tape.

12. Remove the comb by slowly pulling straight up. Do thiscarefully and evenly to prevent tearing the sample wells.

13. Place the gel (on its bed) into the electrophoresis chamber, properlyoriented, centered and level on the platform.

14. Fill the electrophoresis apparatus chamber with the required vol-ume of diluted buffer (see guidelines presented in Table B).

Concentrated Distilled Total Buffer (50x) + Water = Volume

EDVOTEKModel #

M6

M6 +

M12, M20

M36

4 ml 196 ml 200 ml

6 ml 294 ml 300 ml

8 ml 392 ml 400 ml

10 ml 490 ml 500 ml

Electrophoresis (Chamber) BufferTable B: 15. Make sure the gel is completely coveredwith buffer. The agarose gel is some-times called a "submarine gel" becauseit is submerged under buffer for sampleloading and electrophoretic separation.

16. Load samples in wells and conduct elec-trophoresis according to experiment in-structions starting on page 16.

16




Conducting Agarose Gel Electrophoresis

Reminder:

During electrophoresis, the DNAsamples migrate through theagarose gel towards the positiveelectrode. Before loading thesamples, make sure the gel isproperly oriented in the apparatuschamber.

Volts Recommended Time

Minimum Optimal

50 60 min 2.0 hrs

70 40 min 1.5 hrs

125 30 min 45 min

Time and VoltageTable C:

* The EDVOTEK Model #M6 shouldnot be run at higher than 70 volts.

Have a waterbath or beaker of water warmed to 65°C for heating thetubes containing DNA fragments before gel loading. At 65°C, non-specific aggregation due to sticky ends generated by restriction en-zyme digestions will melt. This will result in sharp individual DNAbands upon separation by agarose gel electrophoresis.

LOADING DNA SAMPLES

1. Each group should load 40 µl of the prepared plas-mid DNA sample in a well of the agarose gel.

2. Remember to note the well in which eachgroup loaded its sample.

RUNNING THE GEL

1. After the samples are loaded, carefully snap the cover down ontothe electrode terminals.

Make sure that the negative and positive indicators on the coverand apparatus chamber are properly oriented.

2. Insert the plug of the black wire into the blackinput of the power source (negative input).Insert the plug of the red wire into the redinput of the power source (positive in-put).

3. Set the power source at the required volt-age and run the electrophoresis for the lengthof time as determined by your instructor. General guidelines arepresented in Table C at left.

4. Check to see that current is flowing properly - you should seebubbles forming on the electrodes.

5. Allow the tracking dye to migrate 3.5 to 4 centimeters from thewells for adequate separation of the DNA bands.

6. After the electrophoresis is completed, turn off the power, unplugthe power source, disconnect the leads and remove the cover.

7. Proceed to instructions for staining the gel for DNA Visualizationwith either DNA Blue InstaStain™ or Methylene Blue Plus™ .

+-Black Red

Sample wells

17




Staining & Visualization of DNA

WEAR SAFETY GOGGLESAND GLOVES

Advantages ofDNA Blue InstaStain™

vs.Liquid Staining

• Safe and Simple to Use

• Quick 15-minute staining

• Uniformity of Staining

• Minimal liquid waste

NEWStain DNA with

DNA Blue InstaStain™

Patents Pending

DNA BLUE INSTASTAIN™

EDVOTEK agarose gel electrophoresis experiments now feature a newproprietary staining method for staining DNA. Based on state-of-the-art technology, DNA Blue InstaStain™ is safe, quick, and minimizesthe mess of conventional DNA staining with blue stains.

Staining with DNA Blue InstaStain™

1. After electrophoresis is completed, placethe gel on a flat surface. Moisten the gelwith several drops of electrophoresisbuffer.

2. Wearing gloves, place the blue side ofthe DNA Blue InstaStain sheet on thewell-moistened gel.

3. Firmly run your fingers over the entiresurface of the DNA InstaStain.

Do this several times.

4. Place the gel and DNA Blue InstaStainon a piece of plastic wrap. Then put thegel casting tray and a small empty bea-ker on top.

This will ensure that the InstaStain sheetmaintains good contact with the gel surface.

Allow the DNA Blue InstaStain™ to sitfor 15 minutes.

DNA Blue InstaStain

Patents Pending


Patents Pending

Patents Pending


-----


18




Staining & Visualization ofDNA, cont. Destaining and Visualization of DNA

5. After 15 minutes, remove the sheet of DNABlue InstaStain and transfer the gel to alarge weigh boat or small plastic con-tainer.

6. Conduct destaining with distilled water that has been warmed to37°C.

• First destain: submerge the gel under a smallamount of 37°C distilled water for 15 minuteswith occasional agitation.

• Second destain: submerge the gelunder a small amount of 37°C dis-tilled water for another 15 min-utes with occasional agitation.

7. After the first destain, the largerDNA bands will be visible as darkblue bands against a lighter bluebackground. When completelydestained, the dark blue DNAbands will become clearer and the entire back-ground will becomeuniformly light blue in color.

8. Carefully remove the gel from the destain solu-tion and examine the gel on a Visible Light GelVisualization System. To optimize visibility, usethe amber filter provided with EDVOTEK equip-ment.

9. If the gel is too light and bands are difficult to see, repeat the stain-ing and destaining procedures.

Storage and Disposal of Gel

• A gel stained with DNA Blue InstaStain™ may be stored in therefrigerator for several weeks. Place the gel in a sealable plasticbag with destaining liquid.

DO NOT FREEZE AGAROSE GELS.

• Stained gels which are not kept can be discarded in solid wastedisposal.

DO NOT EXCEED 37°C !Warmer temperatures will

soften the gel and maycause it to break.

A large weigh boat works well fordestaining a 7 x 7 cm gel. Use50 ml of 37°C distilled water tosubmerge the gel in a large weighboat.

( + )

( - )

Useful Hint!


19





WEAR SAFETY GOGGLESAND GLOVES

TRADITIONAL LIQUID STAINING WITHMETHYLENE BLUE PLUS™

1. Remove each gel from its bed and totally submerse the gel(s) inone tray containing 600 ml of diluted Methylene Blue Plus™ stain.

Do not stain gel(s) in the electrophoresis apparatus.

2. Stain gel(s) for a minimum of 30 minutes, with occasional agita-tion.

3. Conduct destaining twice in 600 ml of distilled water that has beenwarmed to 37°C.

• First destain: completely submerse the gel(s) in 600 ml of 37°Cdistilled water for 15 minutes with oc-casional agitation. Then discard thedestaining solution

• Second destain: completely submersethe gel(s) in 600 ml of 37°C distilled wa-ter for another 15 minutes with occa-sional agitation.

Bands will become clearly visible after thesecond destain. You may also leave thegel(s) in destain overnight.

5. Carefully remove the gel from the destain solution and examineon a Visible Light Gel Visualization System. To optimize visibility,use the amber filter provided with EDVOTEK equipment.

6. If the gel is too light and bands are difficult to see, repeat the stain-ing and destaining procedures.

Storage and Disposal of Gel

• Gels stained with Methylene Blue Plus™ may be stored in therefrigerator for several weeks. Place the gel in a sealable plasticbag with destaining liquid.

DO NOT FREEZE AGAROSE GELS.

• Stained gels which are not kept can be discarded in solid wastedisposal.

DO NOTEXCEED 37°C !

Warmertemperatures will

soften the geland may cause it

to break.

Staining & Visualization ofDNA, cont.

Remember!

Dilution of Methylene BluePlus™ stain:

Dilute the 10x stain by mixing 1 partstain with 9 parts distilled ordeionized water.

20




Study Questions

1. What are some reasons for isolating plasmid DNA?

2. What are the functions of sodium hydroxide and SDS in the celllysis solution? What is the function of the potassium acetate solu-tion?

3. What structural property of plasmid DNA allows it to be sepa-rated from chromosomal DNA during alkaline cell lysis?

4. Was more than one band observed in your plasmid sample afterelectrophoresis and staining?

31




Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)

EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

®

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

50x Electrophoresis Buffer

This product contains no hazardous materials as defined by the OSHA HazardCommunication Standard.

No data

No data

No data

No data

No data

No data

Appreciable, (greater than 10%)

Clear, liquid, slight vinegar odor

No data

N.D. = No data

N.D. N.D.

Use extinguishing media appropriate for surrounding fire.

Wear protective equipment and SCBA with full facepieceoperated in positive pressure mode.

None identified

09/15/97

Stability

Section V - Reactivity DataUnstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

X None

Strong oxidizing agents

Carbon monoxide, Carbon dioxide

X None

Yes Yes Yes

None

None identified

Irritation to upper respiratory tract, skin, eyes

None

Ingestion: If conscious, give large amounts of water

Eyes: Flush with water Inhalation: Move to fresh air Skin: Wash with soap and water

Wear suitable protective clothing. Mop up spill

and rinse with water, or collect in absorptive material and dispose of the absorptive material.

Dispose in accordance with all applicable federal, state, and local enviromental regulations.

Avoid eye and skin contact.

None

Yes None

Yes None

Yes Safety goggles

None

None








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Tris-EDTA Buffer (TE)

8-8-98

CAS # 139-33-3 ------------------- No data ----------------

No data

No data

No data

No data

No data

No data

Soluble

Clear, no odor

No data

Dry chemical, carbon dioxide, halon, water spray or standard foam

Thermal decomposition products may include toxic and hazardous oxides of carbon, nitrogen,and sodium.

Move container from fire area if possible

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


Renal or heart disease, potassium deficiency, insulin dependent, diabetes, seizures or intracranial lesions.

X Excessive heat, sparks or open flame

X

Impervious clothing to prevent skin contact

Emergency eye wash should be available

Acids, aluminum, metals, oxidizers (strong)

Yes Yes Yes

Mucous membrane irritation, eye/skin irritation, irritating to gastrointestinal system.

Treat symptomatically and supportively

Mop up with absorptive material. Containerize to dispose or properly

Observe federal, state, and local laws.

Stores away from strong oxidizers or heat. Avoid skin/eye contact.

None

Yes None

Vent. Sys. None

Yes Splash proof goggles

Thermal decomposition products of toxic and hazardous oxides of C, N, & Na

None

Moderately toxic by ingestion. Systemic toxicity may result.

None No data No data No data

Chemical cartridge respirator with full facepiece and organic vapor cartridge

May chelate lead magnesium, zinc, trace metals if present in intestine poss. causing incr.absorption.








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Agarose

8/25/97

This product contains no hazardous materials as defined by the OSHA Hazard CommunicationStandard.CAS #9012-36-6

For 1% solution 194° F

No data

No data

No data

No data

No data

Insoluble - cold

White powder, no odor

N.D. = No data

No data N.D. N.D.

Water spray, dry chemical, carbon dioxide, halon or standard foam

Possible fire hazard when exposed to heat or flame

None

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions



Mechanical (General)Gen. dilution ventilation


Protective Gloves



Eye Protection



Impervious clothing to prevent skin contact

None

X None

No data available

X None

Yes Yes Yes

Inhalation: No data available Ingestion: Large amounts may cause diarrhea

No data available

No data available


Sweep up and place in suitable container for disposal

Normal solid waste disposal

None

None

Chemical cartridge respirator with full facepiece.








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



D/202 Potassium Acetate

5/22/98

Potassium Acetate No data No data No data No dataC2H3KO2

No data

No data

No data

No data

No data

No data

200% at 20°C

Clear liquid, vinegar-like odor

No data No data No data

Dry chemical, carbon dioxide, water spray or foam

Move container from fire area if possible. Avoid breathing vapors

None

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None

None

Thermal decomposition may release smoke and irritating fumes.

X None

Moderately toxic by ingestion

No data

Not required

Avoid contact

Yes Yes Yes

May cause skin/eye irritation. May cause nausea, sore throat, coughing, and abdominal pain

Unknown

Induce vomiting if ingested. For skin/eye contact, flush with large amounts of water.

Mop up with absorbant material and dispose of properly.

Follow all federal, state, and local regulations.

Wear eye protection

None

SCBA with full facepiece

No None

Gen. dilution vent. None

None Splash proof goggles








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Practice Gel Loading Solution

8/25/97

This product contains no hazardous materials as defined by the OSHA Hazard CommunicationStandard.

No data

No data

No data

No data

No data

No data

Soluble

Blue liquid, no odor

No dataNo data No data

Dry chemical, carbon dioxide, water spray or foam

Use agents suitable for type of surrounding fire. Keep upwind, avoid

breathing hazardous sulfur oxides and bromides. Wear SCBA.

Unknown

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None

None

Sulfur oxides, and bromides

X None

Yes Yes Yes

Acute eye contact: May cause irritation. No data available for other routes.

No data available

May cause skin or eye irritation

None reported

Treat symptomatically and supportively. Rinse contacted area with copious amounts of water.

Wear eye and skin protection and mop spill area. Rinse with water.

Observe all federal, state, and local regulations.

Avoid eye and skin contact.

None

Yes None

Yes None


None required

Avoid eye and skin contact








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Sodium Dodecyl Sulfate (SDS)

7/28/98

Lauryl Sulfate, Sodium No data No data No data No data

C12H26O4SCAS# 151-21-3

No data

No data

No data

No data

No data

No data

Soluble

Clear liquid, no odor

No data No data No data

Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam

Wear SCBA and protective clothing to prevent contact with skin & eyes.

May emit toxic fumes.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X


Carbon monoxide, carbon dioxide, sulfur oxides

X

May cause irritation to eyes, ears and nose.

No data

Rubber boots

Avoid prolonged or repeated exsposure.

None

None

Yes Yes Yes

Respiratory tract: burning sensisation, coughing, wheezing, laryngitis, shortness of breath, & headache

No data

Flush skin/eyes with large amounts of water. If inhaled, remove to fresh air.

Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up with absorptivematerial and burn in chemical incinerator equipped with an afterburner and scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Strong sensitizers

NIOSH/MSHA approved respirator.

No Chem. fume hood

No None

rubber Splash proof googles








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



E/202 RNAse (DNAse-Free)

12/18/97

Nuclease, riboCAS # 9001-99-4

No data

No data

No data

No data

No data

No data

Soluble

Clear liquid, no odor

No data N.D. N.D.


Wear SCBA and protective clothing to prevent contact with skin and eyes.

None

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None

X

Yes

Avoid contact and inhalation

Yes Yes YesProlonged or repeated exposure may cause allergic reactionin some individuals.

-------------------------- No data ---------------------------

Unknown

Unknown


No NoneYes None

None

None

None

Mop up with absorbant material. Dispose of properly.

Follow all federal, state, and local regulations.

Avoid eye and inhalation.

None

Chemical resistant Splash proof goggles

NIOSH-MSHA approved respirator








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point




01/31/00

Methylene Blue

3.7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride No data availableCAS # 61-73-4

No data

No data

No data

No data

No data

No data

Soluble - cold

Chemical bound to paper, no odor

No data available No data No data


Self contained breathing apparatus and protective clothing to prevent contact with skin and eyes

Emits toxid fumes under fire conditions

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None


Toxic fumes of Carbon monoxide, Carbon dioxide, nitrogen oxides, sulfur oxides, hydrogen, chloride gas

X None

Yes Yes Yes

Skin: May cause skin irritation Eyes: May cause eye irritation Inhalation: Cyanosis

Meets criteria for proposed OSHA medical records rule PEREAC 47.30420.82

No data available

No data available

Treat symptomatically

Ventilate area and wash spill site

Mix material with a combustible solvent and burn in chemical

incinerator equipped with afterburner and scrubber. Check local and state regulations.

Keep tightly closed. Store in cool, dry place

None

MIOSH/OSHA approved, SCBA

Required

Rubber Chem. safety goggles

Rubber boots








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Sodium Hydroxide

Sodium Hydroxide 2mg/m3 2mg/m3 No dataCAS # 1310-73-2

11/4/98

1390°C

20°C

NO data

2.13

318°C

NO data

NA NA NA

Use extinquishing media appropriate for surrounding fire

Wear protective equipment and self-contained breathing apparatus. Floof material with water

Contact with moisture or water generate sufficient heat to ignite other materials.React with metals to produce hydrogen gas which can form explosive mixture with air.

10% appreciable

White pellets, odorless

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X Moisture

Water, strong acids, metals, combustible materials, organic materialsZinc, aluminous, peroxide, halogenated hydroca

X

Yes Yes Yes

None identified

None No data No data No data

NIOSH/MSHA approved respiratorYes

Yes None

No

Neoprene gloves Safety goggles

Uniform, apron

Avoid contact

None identified

Ingestion: Severe burns to mouth, throat, and stomach, nausea & vomitingInhalation: irritation Skin/eye contact: severe irritation or burns

None identified

Call physician. Ingestion: Do not induce vomiting. Give water followed by vinegar, juice or egg whiteInhalation: Move to fresh air. Skin/eye contact: flush with water

Wear SCBA and protective clothing. Carefully place material into clean, dry container and cover.Dispose of properly

Follow all federal, state, and local laws.

Keep container tightly closed. Store in corrosion-proof area. Store in a dry area.Isolate from incompatible materials.

None

Documents

Mini-prep Isolation of Plasmid DNA - Santa Monica Collegehomepage.smc.edu/bober_mary/Bio 22/Mini-Prep plasmid DNA 202.pdf · Mini-prep EDVO-Kit # Isolation of Plasmid DNA EVT 001171K