Transformation of E. coli with Green and Blue ... - Frederiksen

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222Bacterial TransformationBioluminescence of marine microorganisms has been observed by manysummer visitors at beaches around the world. Onlookers are alwaysfascinated by the repeated parade of color and light on the sand duringthe ebb and flow of the tide. This observation pales to the lightproduced by the bioluminescent jelly fish Aquorea victoria, the naturalhost of the green fluorescent protein (GFP). A bright burst of light isobserved when energy is transferred to the green fluorescent protein(GFP) located in specialized photogenic cells in the base of the jellyfishumbrella. An excellent companion to GFP is the genetically engineeredand well-characterized blue fluorescent protein (BFP).This family of proteins has been known for some time and significantresearch in this area has been reported. Fluorescent proteins can be expressedboth in prokaryotic and eucaryotic cells. These proteins do notrequire substrates, other gene products, or cofactors. When exposed tolong wave U.V. light, they emit a bright green or blue light that is visiblein bacteria transformed by plasmids that contain the genes encodingGFP or BFP. Likewise, purification of the GFP or BFP from crude proteinextracts is simplified by their fluorescence.Background InformationIn cell biology and molecular biology experiments, the GFP protein isoften fused to other proteins to study various biochemical processes.There are many examples of chimeric fusion proteins using the GFP proteinas a biological tag. Such fusions are either at the N- or C- termini.The chimeric proteins are used as biotechnological tools to study proteinlocalization and trafficking within cells.The green fluorescent protein (GFP) possesses a molecular weight ofapproximately 40,000 daltons. Most of the intact protein is requiredfor maintaining fluorescence; only small deletions of a few amino acidsare allowed to be altered without compromising the integrity of theprotein structure. Interestingly, the chromophore responsible for lightemission is within the structure of the GFP protein and resides in aminoacid residues 65 to 67, a cyclic tripeptide composed of Ser-Tyr-Gly. Theimportance of protein folding is clearly demonstrated in that the GFP isfluorescent only upon proper conformational folding.The blue fluorescent protein (BFP) is a variant of the GFP with a His toTyr substitution at position 66 and a second substitution from Tyr to Pheat position 145. With the crystal structure of GFP determined, severalother variants of the GFP have been constructed using site-directed mutagenesis(SDM). SDM allows specific (point) mutations to be introducedin a protein to determine the impact of that mutation on the proteinstructure and function. The set of GFP and BFP proteins can also be usedas a dramatic tool to visually demonstrate the effect of pivotal aminoacid changes on the structure and function of a protein. This, in turn,Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsBacterial TransformationBackground Informationcan be used to help students to appreciate genetically inherited diseasesand the effects of accumulated mutations on aging and the onset ofvarious diseases.Bacterial transformation and Overexpression ofTransformed GenesBacterial transformation is of central importance in molecular biology.Transformation is the process by which a bacterium takes up and expressesexogenous DNA, resulting in a newly acquired genetic trait thatis stable and heritable. This exogenous DNA can be recombinant DNAmolecules that have been constructed in vitro, as well as natural DNAmolecules. Transformation is also of historical importance since it led tothe discovery by Oswald Avery, in 1944, that DNA was the genetic material.In that historical experiment, Avery and colleagues purified DNAfrom a lethal strain of Streptococcus pneumoniae, removing all proteinfrom the DNA. This DNA was then transformed into a harmless strainof the same organism. Injection of the transformed, formerly harmless,strain into mice resulted in their death.For transformation to occur, bacterial cells must be in a particular physiologicalstate, referred to as competency, in which the bacterial cellwall is made permeable to macromolecules such as DNA. Competencycan occur naturally in certain species of Haemophilus and Bacillus whenthe levels of nutrients and oxygen are low. Competent Haemophiluscells express a membrane-associated transport complex that binds andtransfers certain DNA molecules from the medium into bacterial cellsthat are then integrated into the bacterial chromosome and expressed.In nature, the source of the external DNA is from other cells that havedied and their cell walls lysed to release their DNA into the surroundingmedium.Much current research in molecular biology involves the transformationof E. coli, an organism that does not naturally enter a state of competency.E. coli can artificially be made competent when treated withchloride salts of the metal cations calcium, magnesium and rubidium.In addition, abrupt transitioning between heat and cold can induce competency.It is believed that metal ions and temperature changes affectthe structure and permeability of the cell wall and membrane, allowingDNA molecules to pass through the bacterial cell wall. Due to theirunstable cell walls, competent E. coli cells are fragile and therefore mustbe treated carefully.The number of cells transformed per 1 microgram (µg) of DNA is knownas the transformation efficiency. In practice, much smaller amounts ofDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222Bacterial TransformationNumber oftransformantsµg of DNA100transformants0.01 µgXSpecific example:XDNA are used (5 to 100 nanograms, ng) since excessive DNA (>100 ng)inhibits the transformation process. For example, when 10 nanograms(0.01 microgram) of DNA was usedto transform cells that were in a finalfinal vol atrecovery (ml)vol plated (ml)1 ml0.1 mlFigure 1:Bacterial Transformation Efficiency Calculation==Number oftransformantsper µg100,000(1 x 10 5 )transformantsper µgvolume of 1 ml, and if 0.1 ml (100 µl)of these cells were plated on mediumthat contained antibiotic, only thecells that acquired the foreign DNAcould grow. This procedure is calledselection. After incubation (in thisexample) 100 colonies were found onthe plate. Realizing that each colonyoriginally grew from one transformedcell, the transformation efficiency inthis example is 10 5 (outlined in Figure1). In research laboratories, transformationefficiencies generally rangefrom 1 x 10 7 to 1 x 10 8 cells per microgramof DNA. Special procedures canproduce cells having transformationefficiencies approaching 10 10 .The ExperimentTransformation is never 100% efficient. Approximately one in every10,000 cells (of average competency) successfully incorporates exogenousDNA. However, based on the large number of cells (typically 1x 10 9 ), only a small number of cells are transformed to obtain visiblecolonies on agar plates.This concept can be demonstrated by plating the same volume of recoveredcells on selective and nonselective agar medium. The nonselectivemedium will have many more growing cells since all the untransformedcells survive, in addition to the transformed cells. The nonselectivebacterial agar plates will be covered heavily with untransformed cells,forming a “lawn”, in contrast to individual colonies obtained on theselective agar plate.To ferry foreign genes into bacteria, plasmids are usually used. Plasmidsare self-replicating extrachromosomal, double-stranded circular DNAmolecules found in many strains of bacteria. Many plasmids containgenes that provide resistance to various antibiotics, including tetracycline,kanamycin, and ampicillin (amp). Ampicillin is a derivative ofpenicillin that inhibits bacterial growth by interfering with the synthesisof bacterial cell walls. The product of the ampicillin resistance gene isthe enzyme b-lactamase. This enzyme is secreted by transformed cellsinto the surrounding medium, where it destroys ampicillin. Due tothis extracellular secretion, cells that are not transformed are able toundergo limited growth in the zones surrounding transformed, antibiotic-resistantcells. Colonies consisting of these untransformed cellsDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsBacterial TransformationBackground InformationSupercoiledRelaxedFigure 2: Supercoiled andcircular forms of plasmid DNAsare called "satellites", since they only appear around larger colonies oftransformed cells. Larger plating volumes and longer incubation timesincrease the number of satellite colonies.Plasmids naturally exist as supercoiled molecules. The twostrands of DNA in the supercoiled molecule wind around eachother to produce a condensed, entangled structure when comparedto relaxed (non-supercoiled) DNA (Figure 2). CompetentE. coli cells are sensitive to the conformation of the DNA theywill accept. Supercoiled DNA gives the highest transformationefficiencies.SupercoiledpFluoroGreenDNABacterialchromosomal DNAGreenfluorescentproteinFigure 3Overview of GFP and BFP ExpressionIn this experiment, the goal is to express fluorescent proteins (GFP andBFP) in transformed bacterial cells. To begin this process, there must bea means of "turning on" the cloned GFP or BFP gene in the recombinantplasmid. In order to have an "off/on" switch for controlling expression,the gene is placed under the control of a DNA sequence known as a"promoter".A promoter is a sequence in DNA that typically occurs just in front("upstream") of the DNA coding sequence (the sequence that specifiesthe amino acid sequence for a protein). The chromosome of the hostbacterial strain used in this experiment has been genetically engineeredto contain the gene for RNA polymerase, which is under control of thelac promoter, and can be turned on (induced) by the presence of a smallmolecule called IPTG (isopropyl-beta-D-thiogalactopyranoside). IPTGbinds to and inactivates an inhibitor protein known as the lac repressor.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222Bacterial TransformationThe sequence of events required to turn on expression of GFP/BFP is asfollows:1. Cells are grown in the presence of IPTG (to turn on the lac promoter)which binds to, and releases the lac repressor that sits on apromoter (lac) upstream of the RNA polymerase gene. The releaseof the inhibitor allows the RNA polymerase to be produced from theE. coli genome.2. The RNA polymerase, in turn, recognizes the promoter on theplasmid mRNA enabling production of large quantities of thefluorescent protein. In summary, a strong promoter, combined withan active RNA polymerase, allows for very high levels of GFP or BFPmRNA (and thus protein) expression in the transformed cells.Background InformationDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

10EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsExperiment OverviewBefore you start the experiment1. Read all instructions before starting the experiment.The Experiment2. Write a hypothesis that reflects the experiment and predict experimentaloutcomes.Experiment Objective:The objective of this experiment module is to develop an understandingof the biological process of bacterial transformation by pFluoroGreenor pFluoroBlue DNA. This experiment enables the students to observethe acquired phenotypic trait of green or blue fluorescent protein exhibitedby transformed bacterial cells.Brief Description of Experiment:In this experiment, you will transform a strain of competent E. coli whichhas no antibiotic resistance with supercoiled plasmid DNA which has agene for antibiotic resistance. Two plasmids are used for transformation.The plasmid that produces the green fluorescent protein containingthe GFP gene is pFluoroGreen. The plasmid producing the bluefluorescent protein is pFluoroBlue. Both pFluoroGreen and pFluoroBluealso possess the gene for ampicillin (amp) resistance.Bacterial cells will be selected for the presence of plasmid by platingthem on agar medium containing ampicillin. Only bacterial cells thattake up the plasmids will survive selection on ampicillin agar plates.When using pFluoroGreen, green fluorescent colonies will be visibleunder long U.V. Light. Likewise, pFluoroBlue will be visualized asfluorescent blue colonies. If both plasmids are used in a single transformation,then a mixture of green and blue fluorescent colonies willbe visible. The transformation efficiency will then be estimated. Thefluorescent blue colonies will not be as highly fluorescent as the greencolonies.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 11Experiment OverviewStreak E. coli hostcells for isolationDAY BEFORE LABPrepare 5Source PlatesE. coli Cells- DNAPlasmid DNATransfer 2-4 large colonies to eachtube and completely resuspend+ DNA+pFluoroGreen and/orpFluoroBlue10 µlThe Experiment250 µlCaCl 2Incubate on ice for15 minutesIncubate at 42°Cfor 90 secondsIncubate on icefor 2 minutes- DNAAdd 250 µlLuria Recovery Broth37°C for30 minutes+ DNAControlExperimentLB-LB/Amp - LB+IncubateIncubateLB/Amp +overnight in 37°Cincubation ovenovernight in 37°Cincubation ovenLB- LB/Amp - LB+ LB/Amp +LONG WAVE U.V. LIGHTIS REQUIREDTO OBSERVEFLUORESCENT COLONIES.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

12EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsLaboratory SafetyImportant READ ME!The ExperimentTransformation experiments contain antibiotics which are used for theselection of transformed bacteria. Students who have allergies to antibioticssuch as penicilllin, ampicillin, kanamycin or tetracycine shouldnot participate in this experiment.1. Gloves and goggles should be worn routinely as good laboratorypractice.2. Exercise extreme caution when working with equipment which isused in conjunction with the heating and/or melting of reagents.3. Do not mouth pipet reagents - use pipet pumps or bulbs.4. The E. coli bacteria used in this experiment is not considered pathogenic.Although it is rarely associated with any illness in healthyindividuals, it is good practice to follow simple safety guidelines inhandling and disposal of materials contaminated with bacteria.5. Properly dispose materials after completing the experiment:A. Wipe down the lab bench with a 10% bleach solution or a laboratorydisinfectant.B. All materials, including petri plates, pipets, transfer pipets,loops and tubes, that come in contact with bacteria should bedisinfected before disposal in the garbage. Disinfect materialsas soon as possible after use in one of the following ways:• Autoclave at 121° C for 20 minutes.Tape several petri plates together and close tube caps beforedisposal. Collect all contaminated materials in an autoclavable,disposable bag. Seal the bag and place it in a metal tray to preventany possibility of liquid medium or agar from spilling intothe sterilizer chamber.• Soak in 10% bleach solution.Immerse petri plates, open tubes and other contaminated materialsinto a tub containing a 10% bleach solution. Soak the materialsovernight and then discard. Wear gloves and goggleswhen working with bleach.6. Wear gloves, and at the end of the experiment, wash hands thoroughlywith soap and water.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 13Transformation of E.coliSetting up the Transformation and ControlExperimentReminder“+”“-”- DNAContains oneor both plasmidDNAs+ DNADoes not containplasmid DNA1. Label one microcentrifuge tube "+ DNA". This will be thetransformation tube with one or both plasmid DNAs.2. Label a second microcentrifuge tube "– DNA" . This will bethe experimental control tube without plasmid DNA.3. Using a sterile 1 ml pipet, add 250 µl (0.25 ml) of ice cold CaCl 2solutionto each tube.4. As done in research laboratories, pick colonies from the source plateof E. coli cells. Use the following method for each of the test tubeslabeled "+ DNA" and "– DNA":• Use a sterile toothpick to transfer several colonies (2-4 colonies,approximately 2-4 mm in size) from the source plate to the testtube.• Between your fingers, twist the toothpick vigorously and upand down in the cold CaCl 2solution to dislodge the cells.The ExperimentSource PlateE. coli Cells5. In both tubes, suspend the cells completely by tapping or vortexing.6. To the tube labeled "+ DNA", add one of the following options:• 10 µl of pFluoroGreen(from tube labeled "pFG")• 10 µl of pFluoroBlue(from tube labeled "pFB")- DNATransfer 2-4 largecolonies to eachtube of ice cold+CaCl 210 µl+ DNApFluoroGreen and/orpFluoroBlue• 5 µl of each(a total volume of 10 µl)250 µlCaCl2Avoid scraping up agar when transferring the cellsfrom the source plate to the tubes with calciumchloride solution. It is important that the cells areresuspended in the calcium chloride solution and isnot left on the toothpick or on the wall of the tube.Proper experimental techniqueis critical - the plasmid DNA(pFluoroGreen or pFluoro-Blue) must be added directlyinto the cell suspension.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

14EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsTransformation of E.coli- DNA+ DNA7. Incubate the two tubes onice for 15 minutes.The Experiment8. Place both transformation tubes at42°C for 90 seconds.This heat shock step facilitates theentry of DNA in bacterial cells.- DNA+ DNA9. Return both tubes immediately tothe ice bucket and incubate for two(2) minutes.- DNA+ DNA10. Using a sterile pipet, add 250 µl of LuriaRecovery Broth to each tube and mix.Quick Reference:- DNA+ DNA11. Incubate the cells for 30 minutesin a 37°C waterbath for arecovery period.- DNA+ DNADNA and competent cellsare combined in a suspension.After the cells have incubatedwith the DNA, growthmedium (recovery broth)is added. Bacterial cellscontinue to grow throughthe recovery process, duringwhich time the cell wall isrepaired. Cells recover andbegin to express the antibioticresistance gene.12. While the tubes are incubating, label 4 agarplates as indicated below. Write on the bottomor side of the petri plate.• Label one unstriped plate: LB -• Label one unstriped plate: LB + (pFG, pFB or both plasmids)• Label one striped plate: LB / Amp -• Label one striped plate: LB / Amp + (pFG, pFB or both plasmids)• Put your initials or group number on all the plates.13. After the recovery period, remove the tubesfrom the water bath and place them on thelab bench. Proceed to plating the cells forincubation.- DNA+ DNADuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 15Transformation of E.coliReminder“+”“-”Contains oneor both plasmidDNAsDoes not containplasmid DNAPlating the cellsPlating cells from the tube labeled "- DNA" (Control Experiment):14. Use a sterile 1 ml pipet to transfer recovered cells from the tubelabeled " - " to the middle of the following plates:• 0.25 ml to the plate labeled LB -• 0.25 ml to the plate labeled LB / AMP -(see Figure at left).15. Spread the cells over the entire plate with a sterile inoculating loop16. Cover both plates and allow theliquid to be absorbed.The ExperimentSpread cells inone directionSame plate -spread cells 90°to first directionPlating cells from the tube labeled"+ DNA"To avoid contamination when plating, do notset the lid down on the lab bench -- Lift thelid of the plate only enough to allow spreading.Be careful to avoid gouging the loopinto the agar.17. Use a sterile 1 ml pipet to transfer recovered cells from the tubelabeled "+ DNA" to the middle of the following plates:• 0.25 ml to the plate labeled LB +(pFG, pFB or both plasmids)• 0.25 ml to the plate labeled LB / Amp + (pFG, pFB or both plasmids)18. Spread the cells with a sterile inoculating loop in the same manneras step 15 .Reminder:Follow properproceduresfor disposal ofcontaminatedmaterials.19. Cover the plate and allow the liquid to be absorbed (approximately15-20 minutes).Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

16EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsTransformation of E.coliPreparing Plates for Incubation20. Stack your group's set of plates on top of one another and tapethem together. Put your initials or group number on the taped setof plates.The ExperimentReminder:Follow properproceduresfor disposal ofcontaminatedmaterials.The plates should be left in the upright position to allow the cell suspensionto be absorbed by the agar.21. Place the set of plates in a safe place designated by your instructor.22. After the cell suspension is absorbed by the agar,you or your instructor will place the plates in theinverted position (agar side on top) in a 37°Cbacterial incubation oven for overnight incubation(15-20 hours).If the cells have not been absorbed into the medium,it is best to store incubate the plates upright.The plates are inverted to prevent condensation onthe lid, which could drip onto the culture and mayinterfere with experimental results.Group 4Viewing Plates After Incubation23. Darken the room and use a long wave U.V. light to visualize thetransformed cells that will glow as green or blue due to the expressionof the green or blue fluorescent proteins.To visualize the fluorescent colonies, the long wave U.V. light (ED-VOTEK cat. # 969 recommended) can be held underneath the plates ina darkened room.24. Proceed to analyzing your results.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 17Experiment Results and AnalysisLaboratory notebook recordings:Address and record the following in your laboratory notebook or on aseparate worksheet.Before starting the Experiment:• Write a hypothesis that reflects the experiment.• Predict experimental outcomes.During the Experiment:• Record (draw) your observations, or photograph the results.Following the Experiment:The Experiment• Formulate an explanation from the results.• Determine what could be changed in the experiment if theexperiment were repeated.• Write a hypothesis that would reflect this change.Answer these questions BEFORE analyzing yourresults.1. On which plate(s) would you expect to find bacteria most like theoriginal non-transformed E. coli cells? Explain.2. On which plate(s) would you find only genetically transformed bacterialcells? Explain.3. Why would one compare plates LB/amp- and LB/amp+?ContinuedDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

18EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsExperiment Results and AnalysisData Collection5. Observe the results you obtained on your transformation and controlplates.The ExperimentTransformation Plates: + DNA• LB +• LB/Amp +Control Plates: - DNA• LB -• LB/Amp -6. Draw and describe what you observe. For each of the plates, recordthe following:• How much bacterial growth do you observe? Determine acount.• What color are the bacteria?• Why do different members of your class have different transformationefficiency values?• If you did not get any results, what factors could be attributedto this fact?Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 19Experiment Results and AnalysisDetermination of Transformation EfficiencyTransformation efficiency is a quantitative determination of how manycells were transformed per 1 µg of plasmid DNA. In essence, it is an indicatorof how well the transformation experiment worked.You will calculate the transformation efficiency from the data you collectfrom your experiment.1. Count the number of colonies on the plate with ampicillin that islabeled:LB / Amp + (Green, Blue or both)A convenient method to keep track of counted colonies is to markthe colony with a lab marking pen on the outside of the plate.The Experiment2. Determine the transformation efficiency using the formula:Number oftransformantsµg of DNAfinal vol atrecovery (ml)x =vol plated (ml)Number oftransformantsper µgExample:Assume you observed 40 colonies:40transformants0.05 µgx0.5 ml0.25 ml=1600(1.6 x 10 3 )transformantsper µgQuick Reference for Expt. 22250 ng (0.05 mg) of DNA is used.The final volume at recovery is 0.50 ml.The volume plated is0.25 ml.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

20EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsStudy QuestionsAnswer the following study questions in your laboratory notebook or ona separate worksheet.The Experiment1. Exogenous DNA does not passively enter E. coli cells that are notcompetent. What treatment do cells require to be competent?2. Why did the recovery broth used in this experiment not containampicillin?3. What is the difference in the amino acid structure of the green andblue fluorescent proteins?4. What evidence do you have that transformation was or was not successful?5. What are some reasons why transformation may not be successful?6. What is the source of the fluorescence?Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 21Instructor’s GuideNotes to the Instructor:Online Orderingnow availableVisit our web site forinformation aboutEDVOTEK's completeline of experiments forbiotechnology andbiology education.EDVO-TECH SERVICEMon - Fri1-800-EDVOTEK(1-800-338-6835)9 am - 6 pm ETImportant READ ME!Transformation experiments contain antibiotics which are used for theselection of transformed bacteria. Students who have allergies to antibioticssuch as penicilllin, ampicillin, kanamycin or tetracycine shouldnot participate in this experiment.Organizing and Implementing the ExperimentClass size, length of laboratory sessions, and availability of equipmentare factors which must be considered in the planning and the implementationof this experiment with your students.The guidelines that are presented in this manual are based on ten laboratorygroups consisting of two, or up to four students. The followingare implementation guidelines, which can be adapted to fit your specificset of circumstances. If you do not find the answers to your questionsin this section, a variety of resources are available at the EDVOTEK website. In addition, Technical Service is available from 9:00 am to 6:00 pm,Eastern time zone. Call 1-800-EDVOTEK for help from our knowledgeabletechnical staff.Technical ServiceDepartmentMon - Fri9:00 am to 6:00 pm ETFAX: (301) 340-0582web: www.edvotek.comemail: edvotek@aol.comPlease have the following information:• The experiment number and title• Kit Lot number on box or tube• The literature version number(in lower right corner)• Approximate purchase dateDay 1: (Prior to the Lab)• Prepare agar plates• Prepare E. coli Cells (overnight incubation).• Dispense the DNA and control bufferDay 2: (Day of Lab Experiment)• Equilibrate water baths at 37°C and42°C; incubation oven at 37°C• Students transform cells and plate forovernight incubation.Day 3: (Day after Lab Experiment)• Students observe transformants andcontrols• Students calculate transformation efficiency• Follow clean up and disposal proceduresas outlined in the Laboratory Safety section.EDVOTEK - The Biotechnology Education Company ®1-800-EDVOTEK • www.edvotek.com24-hour FAX: (301) 340-0582 • email: edvotek@aol.comEVT 005077K

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 23Pre-Lab PreparationsPour Agar Plates(Prior to the Lab experiment)Wear Hot Glovesand Goggles during allsteps involving heating.• For optimal results, prepare plates two days prior to plating and setaside the plates inverted at room temperature.• If they are poured more than two days before use, they should bestored inverted in the refrigerator. Remove the plates from therefrigerator and store inverted for two days at room temperaturebefore use.Heat the ReadyPour Medium1. Equilibrate a water bath at 60°C for step 5 below.2. Loosen, but do not remove, the cap on the ReadyPour mediumbottle to allow for the venting of steam during heating.Instructor’s GuideCaution: Failure to loosen the cap prior to heating or microwavingmay cause the ReadyPour medium bottle to break or explode.3. Squeeze and vigorously shake the plastic bottle to break up thesolid agar into chunks4. Heat the bottle of ReadyPour medium by one of the methods outlinedbelow. When completely melted, the amber-colored solutionshould appear free of small particles.A. Microwave method:• Heat the bottle on High for two 30 second intervals.• Using a hot glove, swirl and heat on High for an additional 25seconds, or until all the ReadyPour medium is dissolved.• Using a hot glove, occasionally swirl to expedite melting.B. Hot plate or burner method:• Place the bottle in a beaker partially filled with water.• Heat the beaker to boiling over a hot plate or burner.• Using a hot glove, occasionally swirl to expedite melting.5. Allow the melted ReadyPour medium to cool. Placing the bottle ina 60°C water bath will allow the agar to cool, whilepreventing it from prematurely solidifying.When the ReadyPour medium reaches approximately60°C, the bottle will be warm to the touch but not burninghot.60˚CDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

24EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsPre-Lab PreparationsLabel (“Stripe”) the Plates6. Use a lab marker to "stripe" the sides of twenty (20) 60 x15 mmpetri dishes. This will provide an easy method of differentiatingbetween plates with ampicillin and plates without ampicillin.Instructor’s Guide• Open one sleeve of 20 plates and stack the plates neatly.• Start the marker at the bottom of the stack and move themarker vertically to the top plate to "stripe" the sides of the 20plates.• These plates will be used for medium with ampicillin.• Do not stripe the second sleeve of plates. These will be thecontrol plates.Pour the PlatesNote: The single bottle of agar medium will be used to make the 5 sourceplates, 20 control plates and 20 Amp plates.7. Pour 5 large E. Coli source platesUse a 10 ml pipet and pipet pump to pour the 5 large plates, 10 mleach, with the ReadyPour medium without ampicillin.8. Add the IPTG to the cooled Ready Pour medium. Recap the bottleand swirl to mix the IPTG.9. Pour 20 control plates(no ampicillin, no-stripe):Use a fresh 10 ml pipet (or the same pipet from step 7) and pipetpump to pour the 20 control plates, 5 ml each with ReadyPour mediumwithout ampicillin.Quick Reference: Pouring Agar Plates• Use a sterile 10 ml pipet with a pipet pump to transfer thedesignated volume of medium to each petri plate. Pipet carefullyto avoid forming bubbles.• Rock the petri plate back and forth to obtain full coverage.• If the molten medium contains bubbles, they can be removed bypassing a flame across the surface of the medium.• Cover the petri plate and allow the medium to solidify.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 25Pre-Lab PreparationsAdd reagents to mediumwhich has beencooled. Hot mediumwill cause reagents,such as ampicillinand IPTG, to rapidlydecompose.Reminder:Follow properproceduresfor disposal ofcontaminatedmaterials.10. Add the entire amount of ampicillin powder to the remaining moltenReadyPour medium in the bottle.11. Recap the bottle and swirl to completely mix the ampicillin.12. Pour 20 transformation plates(with ampicillin, striped plates):Use a fresh 10 ml pipet to pour the twenty (20) striped plates, 5 mleach.13. Allow the agar to cool and resolidify.Note: If plates will be used within two days, store in a sealable plasticbag so the plates will not dry out. Store at room temperature, inverted.If you have extra sterile petri plates on hand, use any remaining mediumto pour additional plates for the optional activity described onSummary of Poured Plates:5 source plates - large plates:10 ml each - ReadyPour mediumInstructor’s Guide20 control plates - small no stripeplates: 5 ml each - ReadyPour mediumwith IPTG (no ampicillin)20 transformation plates - small stripedplates: 5 ml each - ReadyPour mediumwith IPTG and ampicillinDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

26EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsPre-Lab PreparationsDay before the LabThis experiment requires preparation of isolated E.coli host transformationcolonies 16-24 hours before the laboratory experiment, so planaccordingly.Instructor’s GuideImportant: Do not prepare source plates more than 24 hours beforethe experiment. Old source plates will compromise the success of thetransformation experiment.E.coliHost cellsStreak cellsfor isolationonto sourceplates1. With a sterile loop, streak a small clumpof E.coli cells from the slant and streak forisolation onto a source plate (see figures atleft).Do not use plates containing ampicillin.2. Repeat procedures as described for 4 additionalplates. You will have a total of 5source plates.LB Agar3. Incubate plates inverted overnight (16-24hours) in an incubation oven at 37°C.Incubate Source platesinverted overnight at 37°C.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 27Pre-Lab PreparationsDay of the Lab:1. Dispense 1 ml of CaCl 2into microcentrifuge tubes for each of the 10groups and place on ice.2. Dispense 1.5 ml of Luria Broth Medium ("Recovery broth") intotubes for each of the 10 groups and keep at room temperature.Alternatively, the Luria Broth Medium bottle can be placed at a classroompipeting station for students to share.Preparation of DNA3. Label 10 tubes "pFG" (pFluoroGreen) and 10 tubes "pFB" (pFluoroBlue).Instructor’s Guide4. Place the tube of supercoiled pFluoroGreen and/or pFluoroBlueon ice.5. Before dispensing the DNA, tap the tubes until all the sample is atthe tapered bottom of the tube.6. Using an automatic micropipet, dispense 12 µl of the appropriatesupercoiled DNA to each of the microtest tubes labeled "pFG"(pFluoroGreen) or "pFB" (pFluoroBlue).Note: Students will use 10 µl for the transformation experiment.7. Cap the tubes and place them on ice.Each Group Requires:• Sharing - one of 5 E. coli source plates• 1 tube (1 ml) CaCl 2• 1 tube pFluoroGreen plasmid DNA• 1 tube pFluoro Blue plasmid DNA• 2 striped plates• 2 unstriped plates• 4 sterile 1ml pipets• 2 sterile inoculating loops• 1 tube (1.5ml) "Recovery broth"Classroom Equipment:• Water bath(s)• Incubation OvenDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

28EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsOptional Activity:Do not discard the tubes containing transformed bacteria. After platingan aliquot on selection plates, add an additional 50 µl of recovery brothto the tubes and set them in a rack. Leave on the lab bench overnight.If for some reason, transformants do not grow on the selection plates,the remaining cells can be plated as outlined below:Instructor’s Guide1. Collect the bacterial cell pellet by centrifugation in a microcentrifuge.If a microcentrifuge is not available, let the bacteria collect bygravity and do not disturb.2. Remove all except 50 µl of medium (supernatant, top layer).3. Resuspend the cell pellet in remaining medium.4. Spread the entire contents of the tube on selection medium.5. Incubate the plate as before, 16-24 hours in a 37°C incubation oven.6. Follow proper procedures for disposal of contaminated materials.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 29Experiment Results and AnalysisAnswer these questions BEFORE analyzing yourresults.1. On which plate(s) would you expect to find bacteria most like theoriginal non-transformed E. coli cells? Explain.The bacteria on the plate labeled LB- would be identical to the nontransformedstarter E. coli source plate because they did not haveany plasmid added to them, but were re-plated onto an LB plate.2. On which plate(s) would you find only genetically transformedbacterial cells? Explain.The bacteria growing on the plate labeled LB/amp+ would be thegenetically transformed cells since only those cells that have takenup the plasmid which expresses the ampicillin resistance gene andthe fluorescent gene(s) will survive on the plates which contain ampicillin.Instructor’s Guide3. Why would one compare plates LB/amp- and LB/amp+?Cells not treated with the plasmid will not grow on the plate withampicillin (LB/amp-) because they are not expressing the ampicillinresistance gene. However, cells treated with plasmid(s) will grow onthe LB/amp+ plate because they are expressing the ampicillin resistancegene.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 31Study Questions and Answers1. Exogenous DNA does not passively enter E. coli cells that are notcompetent. What treatment do cells require to be competent?E. coli can be artificially induced to enter competency when they aretreated with the chloride salts of the metal cations calcium, magnesiumand rubidium. In addition, sudden cycles of heat and coldhelp to bring about competency. The metal ions and temperaturechanges affect the structure and permeability of the cell wall andmembrane so that DNA molecules can pass through.2. Why did the recovery broth used in this experiment not containampicillin?The recovery broth did not contain ampicillin in order to give thecells a chance to repair themselves and to express their newly acquiredgenes without an immediate challenge.Instructor’s Guide3. What is the difference in the amino acid structure of the green andblue fluorescent proteins?The difference between the two proteins is the substitution of twoamino acids. The difference in mass is negligible and therefore thetwo proteins will be identical in molecular weights.5. What evidence do you have that transformation was successful?A successful transformation will show colonies on the plate labeledLB/amp+ and should fluoresce under long UV light. An unsuccessfultransformation will not show any colonies on the LB/amp+ plate.6. What are some reasons why transformation may not be successful?Unsuccessful transformations could be the result of many things,including: 1) not adding the plasmid to the host cells in the +DNAtube, or 2) not adding a colony of bacteria to the +DNA tube, and 3)improper timing of the heat shock step.7. What is the source of the fluorescence?The source of fluorescence comes from the protein (GFP/BFP) encodedby the plasmid.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

EDVOTEK®Material Safety Data SheetMay be used to comply with OSHA's Hazard CommunicationStandard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.Section V - Reactivity DataStabilityUnstableStable XIncompatibilityStrong oxidizersConditions to AvoidIncompatiblesIDENTITY (As Used on Label and List)AmpicillinSection IManufacturer's NameEDVOTEK, Inc.Address (Number, Street, City, State, Zip Code)14676 Rothgeb DriveRockville, MD 20850Section II - Hazardous Ingredients/Identify InformationHazardous Components [SpecificChemical Identity; Common Name(s)] OSHA PEL ACGIH TLVAmpicillinCAS# 7177-48-2No dataNote: Blank spaces are not permitted. If any item is notapplicable, or no information is available, the space mustbe marked to indicate that.Emergency Telephone Number(301) 251-5990Telephone Number for information(301) 251-5990Date PreparedSignature of Preparer (optional)07/01/03Other LimitsRecommended% (Optional)Hazardous Decomposition or ByproductsHazardousMay OccurConditions to AvoidPolymerizationWill Not Occur XIncompaticlesSection VI - Health Hazard DataRoute(s) of Entry: Inhalation?Yes Skin? Yes Ingestion? YesHealth Hazards (Acute and Chronic)Carcinogenicity:No dataNTP? IARC Monographs? OSHA Regulation?Signs and Symptoms of ExposureMedical Conditions Generally Aggravated by ExposureToxic oxides of carbon, nitrogen and sulfurSensitizers may result in allergic reactionRepeated exposure may result in sensitization and possibleanaphylactic shock.No dataEmergency First Aid Procedures Ingestion: Allergic symptoms.Eyes/Skin: Flush with water Inhalation: Move to fresh airSection III - Physical/Chemical CharacteristicsBoiling PointVapor Pressure (mm Hg.)Vapor Density (AIR = 1)Solubility in WaterAppearance and OdorSlightly solubleNo dataNo dataNo dataOdorless, white crystaline powderSpecific Gravity (H 0 = 1)2Melting PointEvaporation Rate(Butyl Acetate = 1)No dataNo dataNo dataSection VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled Wear suitable protective clothing. Sweep upand place in suitable container for later disposal. Do not flush spilled material down sink.Waste Disposal Method Observe all federal, state, and local regulationsPrecautions to be Taken in Handling and StoringKeep away from incompatible substancesOther Precautions NoneSection IV - Physical/Chemical Characteristics N.D. = No dataFlash Point (Method Used)Flammable Limits LELNo dataN.D.Extinguishing MediaSpecial Fire Fighting ProceduresUnusual Fire and Explosion HazardsAvoid breathing vapors.Dry chemical, carbon dioxide, water spray or regular foamMove container from fire area if possible. Do not scatter spilledmaterial with water streams.UELN.D.Section VIII - Control MeasuresRespiratory Protection (Specify Type)Ventilation Local Exhaust YesSpecialMechanical (General) NoOtherProtective Gloves YesEye ProtectionNoneNoneSplash or dust proofOther Protective Clothing or Equipment Eye washWork/Hygienic PracticesWear protective clothing and equipment to prevent contact.EDVOTEK®IDENTITY (As Used on Label and List)IPTGSection IManufacturer's NameEDVOTEK, Inc.Address (Number, Street, City, State, Zip Code)14676 Rothgeb DriveRockville, MD 20850Material Safety Data SheetMay be used to comply with OSHA's Hazard CommunicationStandard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.Section II - Hazardous Ingredients/Identify InformationHazardous Components [SpecificChemical Identity; Common Name(s)] OSHA PEL ACGIH TLVNot applicableBoiling PointNote: Blank spaces are not permitted. If any item is notapplicable, or no information is available, the space mustbe marked to indicate that.Emergency Telephone Number(301) 251-5990Telephone Number for information(301) 251-5990Date PreparedSection III - Physical/Chemical CharacteristicsVapor Pressure (mm Hg.)Vapor Density (AIR = 1)Solubility in WaterAppearance and OdorSpecial Fire Fighting ProceduresNoneSignature of Preparer (optional)Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)Flammable LimitsUnknownExtinguishing MediaNoneNoneNoneModerateSpecific Gravity (H 0 = 1)2Melting PointEvaporation Rate(Butyl Acetate = 1)07/01/03White crystals/slight odor thiophenolWater, carbon dioxide, or dry chemicalOther LimitsRecommendedLEL% (Optional)Unknown109-110CNoneUELSection V - Reactivity DataStabilityIncompatibilityUnstableStableHazardous Decomposition or ByproductsConditions to AvoidHazardousMay OccurConditions to AvoidPolymerizationWill Not Occur XSection VI - Health Hazard DataRoute(s) of Entry: Inhalation? Skin?Ingestion?Yes Not studied Not studiedHealth Hazards (Acute and Chronic)Toxcity has not been studiedCarcinogenicity: NTP? IARC Monographs? OSHA Regulation?Unknown No data No data No dataSigns and Symptoms of ExposureMedical Conditions Generally Aggravated by ExposureEmergency First Aid ProceduresSection VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for SpilledCover and sweep up with inert carrierWaste Disposal MethodDissolve in a combustible solvent and burn in a chemical incinerator withafterburner and scrubber, or sweep up and return inoriginal container.Precautions to be Taken in Handling and StoringOther PrecautionsSection VIII - Control MeasuresRespiratory Protection (Specify Type)Ventilation Local Exhaust YesSpecialMechanical (General) YesOtherProtective GlovesExternal: flush with waterXAvoid dust store coolInformation CAS #367-93-1Rubber or vinylNoneStrong oxdizing agentsCarbon dioxide and sulfur dioxideUnknown: avoid dustUnknownInternal: Induce vomiting, consult physicianFilter maskEye ProtectionNoneNoneFace mask or gogglesUnusual Fire and Explosion HazardsNoneOther Protective Clothing or EquipmentWork/Hygienic PracticesLab apronAvoid dust or contact with skin