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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
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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
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 ®
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
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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 ®
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
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EDVO-Kit # 202: Mini-prep Isolation of Plasmid DNA
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.
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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 ® 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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EDVO-Kit # 202: Mini-prep Isolation of Plasmid DNA
EDVOTEK • The BiotechnologyEducation Company ®
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
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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 ®
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
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EDVO-Kit # 202: Mini-prep Isolation of Plasmid DNA
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
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 ® EXPERIMENTAL PROCEDURES
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.
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EDVO-Kit # 202: Mini-prep Isolation of Plasmid DNA
EDVOTEK • The BiotechnologyEducation Company ® EXPERIMENTAL PROCEDURES
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.
Wear Safety Goggles & Gloves
Remember!
12
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 ®
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
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 ® EXPERIMENTAL PROCEDURES
Agarose Gel Electrophoresis
Wear Safety Goggles & Gloves
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.
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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 ® EXPERIMENTAL PROCEDURES
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:
Wear Safety Goggles & Gloves
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
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 ®
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.
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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 ® EXPERIMENTAL PROCEDURES
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
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 ®
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
DNA Blue InstaStain™
Patents Pending
Patents Pending
DNA Blue InstaStain™
-----
EXPERIMENTAL PROCEDURES
18
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 ®
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!
EXPERIMENTAL PROCEDURES
19
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 ®
EXPERIMENTAL PROCEDURES
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
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 ® EXPERIMENTAL PROCEDURES
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
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 ®
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
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
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
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
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.
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
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
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)Gen. dilution ventilation
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
Yes Splash proof goggles
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
Treat symptomatically and supportively
Sweep up and place in suitable container for disposal
Normal solid waste disposal
None
None
Chemical cartridge respirator with full facepiece.
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
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
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
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
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
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
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
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
Yes Splash proof goggles
None required
Avoid eye and skin contact
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
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
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
Strong oxidizing agents
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
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
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.
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam
Wear SCBA and protective clothing to prevent contact with skin and eyes.
None
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
X
Yes
Avoid contact and inhalation
Yes Yes YesProlonged or repeated exposure may cause allergic reactionin some individuals.
-------------------------- No data ---------------------------
Unknown
Unknown
Treat symptomatically and supportively
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
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
DNA Blue InstaStain™
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
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam
Self contained breathing apparatus and protective clothing to prevent contact with skin and eyes
Emits toxid fumes under fire conditions
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
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
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
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
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 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