GFPII:Transformation-1

GreenFluorescentProteinII: BacterialTransformationObjectiveTogetsomeexperiencewithrecombinantDNAtechniquesandtogeneticallyalterabacterialstraintoproduceaparticularprotein.BackgroundNotethatwehavenotcoveredthismaterialinlectureyet,so,fortoday,youwillneedtodosomethingswithoutcompletelyunderstandingthem.Evenifallthedetailsarenotclear,thebigpictureisbasedonsomeprettysimpleextensionsofthegeneticsyoualreadyknow.Asthecoursecontinues,whatyouhavedonetodaywillbeclearer.GreenFluorescentProtein(GFP)isaproteinproducedbythejellyfishAequoriavictoria.Theproteinfluorescesgreenwhenexposedtoultraviolet(UV)light.Thegoaloftoday’slabistochangethegenesofabacteriumsothatitnowmakesGFPandwillfluoresceinUVlight.Intoday’slab,youwilladdasmallDNAmoleculecalledaplasmidtoabacteriumcalledE.coli.ThesebacteriaaretheworkhorseofmodernrecombinantDNAtechnology.ThebacteriayouarestartingwithcannotproduceGFP(sincethegeneisnormallyonlyfoundinthejellyfish)andarekilledby(aka“sensitiveto”)theantibioticampicillinTheplasmidyouwillbeaddingiscalled“pGLO”anditcontainstwogenes:

• AmpicillinResistance–thisisagenethatconfersthedominantphenotypeofresistancetotheantibioticampicillin.

• GFP–thisisagenethatproducestheGFPproteininbacteria.YouwilladdthepGLODNAtobacteriathathavebeentreatedtomakethem“competent”–thatis,readytotakeupDNAfromtheenvironment.However,onlyaverysmallfraction(fewerthan1in1,000,000)ofthebacteriawilltakeuptheDNA.ThebacteriathatdotakeupthepGLODNAnowhavethegenesonpGLOaddedtotheirgenome.Theyhavebeen“transformed”.ThesebacteriaarenowresistanttoampicillinandproduceGFP.Wenextselectforthetransformedbacteriabygrowingthecellsinthepresenceofampicillin.Un-transformedcellsaresensitivetoampicillinandarekilled.However,thosethatweretransformedwithpGLOareresistanttoampicillinandwillgrow.Wewillbegrowingourcellsonsolidmedium,soasinglesurvivingcellwillgiverisetoa“colony”of108cells–asmallpileofcells,alldescendantsofthatoriginaltransformedcell.AllthecellsinthecolonywillcarrypGLOandthusbeampicillinresistantandmakeGFP.

GFPII:Transformation-2

ThegeneticmapofpGLOisshownbelow:

Youcanseethegenesforampicillinresistance(AmpR)andGFP;eachofthesegeneshasapromoter.Wewilltalkmoreaboutplasmidsandhowtheyworkinlecture.ProcedureI:Transformation WARNINGS:1. Ingeneral,thelabisunforgivingofmistakeslikeusingthewrongsolutionortakingthewrong

amount.Theconstructionfolksat“ThisOldHouse”,say“Measuretwice;cutonce”.We’lladaptthisto“Checktwice;pipetteonce”.

2. AlthoughtheE.colistrainweuseisnon-pathogenic(itisnotknowntocausediseaseinhealthyindividuals),youshouldbecarefulwithit.Alwaysweargloves,don’teatordrinkinlab,andwashyourhandsthoroughlywhenyouarealldone.

3. SterileTechnique:Contaminationisabigproblem–it’sadirtyworldwelivein.Youshouldassumethatallsurfacesarecrawlingwithnastymicrobes.Neverletanyofthesterilepicks,pipettes,etc.touchanythingexceptthetube,plate,solution,orcolony.Ifyoueventhinkthatyou’vetouchedsomethingyoushouldn’t,discardtheloop,pipette,etcandgetacleanone.

GFPII:Transformation-3

Notethatwhilethefiguresshowplastictransferpipettes,wewillbeusingpipetmen.1)Labeloneclosedmicrotesttube+pGLOandanother-pGLO.Labelbothtubeswithyourgroup’sname.Placetheminthefoamtuberack.2)Openthetubesandusingatransferpipette,transfer250μloftransformationsolution(CaCl2)intoeachtube.3)Placethetubesoncrushedice.Donotusecubedice.4)Useasterilelooptopickupasinglecolonyofbacteriafromyourstarterplate.Pickupthe+pGLOtubeandimmersetheloopintothetransformationsolutionatthebottomofthetube.Spintheloopbetweenyourindexfingerandthumbuntiltheentirecolonyisdispersedinthetransformationsolution(withnofloatingchunks).Placethetubebackinthetuberackintheice.Usinganewsterileloop,repeatforthe-pGLOtube.

18

Transformation Kit—Quick Guide1. Label one closed micro test tube

+pGLO and another -pGLO.Label both tubes with yourgroup’s name. Place them in thefoam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution(CaC12) into each tube.

3. Place the tubes on crushed ice.Do not use cubed ice.

6. Incubate the tubes on ice for10 min. Make sure to push thetubes all the way down in therack so the bottom of the tubesstick out and make contact withthe ice.

5. Examine the pGLO plasmid DNAsolution with the UV lamp. Noteyour observations. Immerse a newsterile loop into the plasmid DNAstock tube. Withdraw a loopful.There should be a film of plasmidsolution across the ring. This issimilar to seeing a soapy filmacross a ring for blowing soapbubbles. Mix the loopful into the cellsuspension of the +pGLO tube.Optionally, pipet 10 µl of pGLOplasmid into the +pGLO tube andmix. Close the -pGLO tube and returnit to the rack on ice. Do not addplasmid DNA to the -pGLO tube.Why not? Close the -pGLGO tubeand return it to the rack on ice.

+p

GLO

-pG

LO

Transformationsolution

-pGLOplasmid DNA

IceRack

-pGLO

Ice

250 µl

+p

GL

O

+p

GL

O

+p

GL

O

-pG

LO

+p

GL

O

-pG

LO

4. Use a sterile loop to pick up a single colony of bacteria from yourstarter plate. Pick up the +pGLOtube and immerse the loop into thetransformation solution at the bottom of the tube. Spin the loopbetween your index finger andthumb until the entire colony is dispersed in the transformationsolution (with no floating chunks).Place the tube back in the tuberack in the ice. Using a new sterileloop, repeat for the -pGLO tube.

QU

ICK

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IDE

18

Transformation Kit—Quick Guide1. Label one closed micro test tube

+pGLO and another -pGLO.Label both tubes with yourgroup’s name. Place them in thefoam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution(CaC12) into each tube.

3. Place the tubes on crushed ice.Do not use cubed ice.

6. Incubate the tubes on ice for10 min. Make sure to push thetubes all the way down in therack so the bottom of the tubesstick out and make contact withthe ice.

5. Examine the pGLO plasmid DNAsolution with the UV lamp. Noteyour observations. Immerse a newsterile loop into the plasmid DNAstock tube. Withdraw a loopful.There should be a film of plasmidsolution across the ring. This issimilar to seeing a soapy filmacross a ring for blowing soapbubbles. Mix the loopful into the cellsuspension of the +pGLO tube.Optionally, pipet 10 µl of pGLOplasmid into the +pGLO tube andmix. Close the -pGLO tube and returnit to the rack on ice. Do not addplasmid DNA to the -pGLO tube.Why not? Close the -pGLGO tubeand return it to the rack on ice.

+p

GL

O

-pG

LO

Transformationsolution

-pGLOplasmid DNA

IceRack

-pGLO

Ice

250 µl

+p

GL

O

+p

GL

O

+p

GL

O

-pG

LO

+p

GL

O

-pG

LO

4. Use a sterile loop to pick up a single colony of bacteria from yourstarter plate. Pick up the +pGLOtube and immerse the loop into thetransformation solution at the bottom of the tube. Spin the loopbetween your index finger andthumb until the entire colony is dispersed in the transformationsolution (with no floating chunks).Place the tube back in the tuberack in the ice. Using a new sterileloop, repeat for the -pGLO tube.

QU

ICK

GU

IDE

18

Transformation Kit—Quick Guide1. Label one closed micro test tube

+pGLO and another -pGLO.Label both tubes with yourgroup’s name. Place them in thefoam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution(CaC12) into each tube.

3. Place the tubes on crushed ice.Do not use cubed ice.

6. Incubate the tubes on ice for10 min. Make sure to push thetubes all the way down in therack so the bottom of the tubesstick out and make contact withthe ice.

5. Examine the pGLO plasmid DNAsolution with the UV lamp. Noteyour observations. Immerse a newsterile loop into the plasmid DNAstock tube. Withdraw a loopful.There should be a film of plasmidsolution across the ring. This issimilar to seeing a soapy filmacross a ring for blowing soapbubbles. Mix the loopful into the cellsuspension of the +pGLO tube.Optionally, pipet 10 µl of pGLOplasmid into the +pGLO tube andmix. Close the -pGLO tube and returnit to the rack on ice. Do not addplasmid DNA to the -pGLO tube.Why not? Close the -pGLGO tubeand return it to the rack on ice.

+p

GL

O

-pG

LO

Transformationsolution

-pGLOplasmid DNA

IceRack

-pGLO

Ice

250 µl

+p

GL

O

+p

GL

O

+p

GL

O

-pG

LO

+p

GL

O

-pG

LO

4. Use a sterile loop to pick up a single colony of bacteria from yourstarter plate. Pick up the +pGLOtube and immerse the loop into thetransformation solution at the bottom of the tube. Spin the loopbetween your index finger andthumb until the entire colony is dispersed in the transformationsolution (with no floating chunks).Place the tube back in the tuberack in the ice. Using a new sterileloop, repeat for the -pGLO tube.

QU

ICK

GU

IDE

18

Transformation Kit—Quick Guide1. Label one closed micro test tube

+pGLO and another -pGLO.Label both tubes with yourgroup’s name. Place them in thefoam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution(CaC12) into each tube.

3. Place the tubes on crushed ice.Do not use cubed ice.

6. Incubate the tubes on ice for10 min. Make sure to push thetubes all the way down in therack so the bottom of the tubesstick out and make contact withthe ice.

5. Examine the pGLO plasmid DNAsolution with the UV lamp. Noteyour observations. Immerse a newsterile loop into the plasmid DNAstock tube. Withdraw a loopful.There should be a film of plasmidsolution across the ring. This issimilar to seeing a soapy filmacross a ring for blowing soapbubbles. Mix the loopful into the cellsuspension of the +pGLO tube.Optionally, pipet 10 µl of pGLOplasmid into the +pGLO tube andmix. Close the -pGLO tube and returnit to the rack on ice. Do not addplasmid DNA to the -pGLO tube.Why not? Close the -pGLGO tubeand return it to the rack on ice.

+p

GL

O

-pG

LO

Transformationsolution

-pGLOplasmid DNA

IceRack

-pGLO

Ice

250 µl

+p

GL

O

+p

GL

O

+p

GL

O

-pG

LO

+p

GL

O

-pG

LO

4. Use a sterile loop to pick up a single colony of bacteria from yourstarter plate. Pick up the +pGLOtube and immerse the loop into thetransformation solution at the bottom of the tube. Spin the loopbetween your index finger andthumb until the entire colony is dispersed in the transformationsolution (with no floating chunks).Place the tube back in the tuberack in the ice. Using a new sterileloop, repeat for the -pGLO tube.

QU

ICK

GU

IDE

GFPII:Transformation-4

5)YourTAwillgiveyouasampleofpGLOplasmidDNAsolution.ExaminethepGLOplasmidDNAsolutionwiththeUVlamp.Noteyourobservations.Usingapipettor,put10μlofpGLOplasmidintothe+pGLOtubeandmix.Closethe+pGLOtubeandreturnittotherackonice.DonotaddplasmidDNAtothe-pGLOtube.Whynot?Closethe-pGLGOtubeandreturnittotherackonice.6)Incubatethetubesonicefor10min.Makesuretopushthetubesallthewaydownintheracksothebottomofthetubesstickoutandmakecontactwiththeice.7)Whilethetubesaresittingonice,labelyourfouragarplatesonthebottom(notthelid)asshownonthediagram.8)Heatshock.Usingthefoamrackasaholder,transferboththe(+)pGLOand(-)pGLOtubesintothewaterbath,setat42°C,forexactly50seconds.Makesuretopushthetubesallthewaydownintheracksothebottomofthetubesstickoutandmakecontactwiththewarmwater.Whenthe50secondshavepassed,placebothtubesbackonice.Forthebesttransformationresults,thechangefromtheice(0°C)to42°Candthenbacktotheicemustberapid.Incubatetubesonicefor2min.9)Removetherackcontainingthetubesfromtheiceandplaceonthebenchtop.OpenthepGLO+tubeand,usinganewsterilepipet,add250μlofLBnutrientbrothtothetubeandrecloseit.RepeatwithanewsterilepipetforthepGLO-tube.Incubatethetubesfor30minatroomtemperature.

18

Transformation Kit—Quick Guide1. Label one closed micro test tube

+pGLO and another -pGLO.Label both tubes with yourgroup’s name. Place them in thefoam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution(CaC12) into each tube.

3. Place the tubes on crushed ice.Do not use cubed ice.

6. Incubate the tubes on ice for10 min. Make sure to push thetubes all the way down in therack so the bottom of the tubesstick out and make contact withthe ice.

5. Examine the pGLO plasmid DNAsolution with the UV lamp. Noteyour observations. Immerse a newsterile loop into the plasmid DNAstock tube. Withdraw a loopful.There should be a film of plasmidsolution across the ring. This issimilar to seeing a soapy filmacross a ring for blowing soapbubbles. Mix the loopful into the cellsuspension of the +pGLO tube.Optionally, pipet 10 µl of pGLOplasmid into the +pGLO tube andmix. Close the -pGLO tube and returnit to the rack on ice. Do not addplasmid DNA to the -pGLO tube.Why not? Close the -pGLGO tubeand return it to the rack on ice.

+p

GL

O

-pG

LO

Transformationsolution

-pGLOplasmid DNA

IceRack

-pGLO

Ice

250 µl

+p

GL

O

+p

GL

O

+p

GL

O

-pG

LO

+p

GL

O

-pG

LO

4. Use a sterile loop to pick up a single colony of bacteria from yourstarter plate. Pick up the +pGLOtube and immerse the loop into thetransformation solution at the bottom of the tube. Spin the loopbetween your index finger andthumb until the entire colony is dispersed in the transformationsolution (with no floating chunks).Place the tube back in the tuberack in the ice. Using a new sterileloop, repeat for the -pGLO tube.

QU

ICK

GU

IDE

18

Transformation Kit—Quick Guide1. Label one closed micro test tube

+pGLO and another -pGLO.Label both tubes with yourgroup’s name. Place them in thefoam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution(CaC12) into each tube.

3. Place the tubes on crushed ice.Do not use cubed ice.

6. Incubate the tubes on ice for10 min. Make sure to push thetubes all the way down in therack so the bottom of the tubesstick out and make contact withthe ice.

5. Examine the pGLO plasmid DNAsolution with the UV lamp. Noteyour observations. Immerse a newsterile loop into the plasmid DNAstock tube. Withdraw a loopful.There should be a film of plasmidsolution across the ring. This issimilar to seeing a soapy filmacross a ring for blowing soapbubbles. Mix the loopful into the cellsuspension of the +pGLO tube.Optionally, pipet 10 µl of pGLOplasmid into the +pGLO tube andmix. Close the -pGLO tube and returnit to the rack on ice. Do not addplasmid DNA to the -pGLO tube.Why not? Close the -pGLGO tubeand return it to the rack on ice.

+p

GL

O

-pG

LO

Transformationsolution

-pGLOplasmid DNA

IceRack

-pGLO

Ice

250 µl

+p

GL

O

+p

GL

O

+p

GL

O

-pG

LO

+p

GL

O

-pG

LO

4. Use a sterile loop to pick up a single colony of bacteria from yourstarter plate. Pick up the +pGLOtube and immerse the loop into thetransformation solution at the bottom of the tube. Spin the loopbetween your index finger andthumb until the entire colony is dispersed in the transformationsolution (with no floating chunks).Place the tube back in the tuberack in the ice. Using a new sterileloop, repeat for the -pGLO tube.

QU

ICK

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IDE

19

10. Gently flick the closed tubes withyour finger to mix. Using a newsterile pipet for each tube,pipet 100 µl from each of thetubes to the correspondingplates, as shown on the diagramonto the appropriate plates.

11. Use a new sterile loop for eachplate. Spread the suspensionsevenly around the surface of theagar by quickly skating the flatsurface of a new sterile loopback and forth across the platesurface.

12. Stack up your plates and tapethem together. Put your groupname and class period on the bottom of the stack and place thestack upside down in the 37°Cincubator until the next day.

7. While the tubes are sitting onice, label your four agar plateson the bottom (not the lid) as shown on the diagram.

8. Heat shock. Using the foam rackas a holder, transfer both the (+)pGLO and (-) pGLO tubes intothe water bath, set at 42°C, forexactly50 seconds. Make sure topush the tubes all the way downin the rack so the bottom of thetubes stick out and make contactwith the warm water. When the50 seconds have passed, placeboth tubes back on ice. For thebest transformation results, thechange from the ice (0°C) to42°C and then back to the icemust be rapid. Incubate tubeson ice for 2 min.

LB-Broth

100 µl

IceIce

Water bath

42°C for 50 sec

9. Remove the rack containing thetubes from the ice and place onthe bench top. Open a tube and,using a new sterile pipet, add250 µl of LB nutrient broth to thetube and reclose it. Repeat witha new sterile pipet for the othertube. Incubate the tubes for 10 min at room temperature.

250 µl

L B/am ppGLO

LB/amp/arapGLO

LB/amppGLO

L B

pGLO

L B

- pG

O

LLB/am p

- pG

O

LLB/am p

+pG

O

LLB/amp/ara

+pG

O

L

+p

GL

O

-pG

LO

QU

ICK

GU

IDE

19

10. Gently flick the closed tubes withyour finger to mix. Using a newsterile pipet for each tube,pipet 100 µl from each of thetubes to the correspondingplates, as shown on the diagramonto the appropriate plates.

11. Use a new sterile loop for eachplate. Spread the suspensionsevenly around the surface of theagar by quickly skating the flatsurface of a new sterile loopback and forth across the platesurface.

12. Stack up your plates and tapethem together. Put your groupname and class period on the bottom of the stack and place thestack upside down in the 37°Cincubator until the next day.

7. While the tubes are sitting onice, label your four agar plateson the bottom (not the lid) as shown on the diagram.

8. Heat shock. Using the foam rackas a holder, transfer both the (+)pGLO and (-) pGLO tubes intothe water bath, set at 42°C, forexactly50 seconds. Make sure topush the tubes all the way downin the rack so the bottom of thetubes stick out and make contactwith the warm water. When the50 seconds have passed, placeboth tubes back on ice. For thebest transformation results, thechange from the ice (0°C) to42°C and then back to the icemust be rapid. Incubate tubeson ice for 2 min.

LB-Broth

100 µl

IceIce

Water bath

42°C for 50 sec

9. Remove the rack containing thetubes from the ice and place onthe bench top. Open a tube and,using a new sterile pipet, add250 µl of LB nutrient broth to thetube and reclose it. Repeat witha new sterile pipet for the othertube. Incubate the tubes for 10 min at room temperature.

250 µl

L B/am ppGLO

LB/amp/arapGLO

LB/amppGLO

L B

pGLO

L B

- pG

O

LLB/am p

- pG

O

LLB/am p

+pG

O

LLB/amp/ara

+pG

O

L

+pG

LO

-pG

LO

QU

ICK

GU

IDE

19

10. Gently flick the closed tubes withyour finger to mix. Using a newsterile pipet for each tube,pipet 100 µl from each of thetubes to the correspondingplates, as shown on the diagramonto the appropriate plates.

11. Use a new sterile loop for eachplate. Spread the suspensionsevenly around the surface of theagar by quickly skating the flatsurface of a new sterile loopback and forth across the platesurface.

12. Stack up your plates and tapethem together. Put your groupname and class period on the bottom of the stack and place thestack upside down in the 37°Cincubator until the next day.

7. While the tubes are sitting onice, label your four agar plateson the bottom (not the lid) as shown on the diagram.

8. Heat shock. Using the foam rackas a holder, transfer both the (+)pGLO and (-) pGLO tubes intothe water bath, set at 42°C, forexactly50 seconds. Make sure topush the tubes all the way downin the rack so the bottom of thetubes stick out and make contactwith the warm water. When the50 seconds have passed, placeboth tubes back on ice. For thebest transformation results, thechange from the ice (0°C) to42°C and then back to the icemust be rapid. Incubate tubeson ice for 2 min.

LB-Broth

100 µl

IceIce

Water bath

42°C for 50 sec

9. Remove the rack containing thetubes from the ice and place onthe bench top. Open a tube and,using a new sterile pipet, add250 µl of LB nutrient broth to thetube and reclose it. Repeat witha new sterile pipet for the othertube. Incubate the tubes for 10 min at room temperature.

250 µl

L B/am ppGLO

LB/amp/arapGLO

LB/amppGLO

L B

pGLO

L B

- pG

O

LLB/am p

- pG

O

LLB/am p

+pG

O

LLB/amp/ara

+pG

O

L

+p

GL

O

-pG

LO

QU

ICK

GU

IDE

GFPII:Transformation-5

10)Gentlyflicktheclosedtubeswithyourfingertomix.Usinganewsterilepipetforeachtube,pipet100ulfromeachofthetubestothecorrespondingplates,asshownonthediagramontotheappropriateplates.Besuretoputthecellsonthejello-likeagarmedium,nottheplasticlid!11)Useanewsterileloopforeachplate.Spreadthesuspensionsevenlyaroundthesurfaceoftheagarbyquicklyskatingtheflatsurfaceofanewsterileloopbackandforthacrosstheplatesurface.12)Stackupyourplatesandtapethemtogether.Putyourgroupnameandclassperiodonthebottomofthestackandplacethestackupsidedowninthe37°Cincubatoruntilthenextday.Onceyourcellshavegrown,yourTAwillputthemintherefrigeratorsoyoucanlookatthemduringthelastweekoflab.LabReportThereisnolabreportforthissession.Youwillwriteuptheresultswhenyouseetheplatesinalaterlab.

19

10. Gently flick the closed tubes withyour finger to mix. Using a newsterile pipet for each tube,pipet 100 µl from each of thetubes to the correspondingplates, as shown on the diagramonto the appropriate plates.

11. Use a new sterile loop for eachplate. Spread the suspensionsevenly around the surface of theagar by quickly skating the flatsurface of a new sterile loopback and forth across the platesurface.

12. Stack up your plates and tapethem together. Put your groupname and class period on the bottom of the stack and place thestack upside down in the 37°Cincubator until the next day.

7. While the tubes are sitting onice, label your four agar plateson the bottom (not the lid) as shown on the diagram.

8. Heat shock. Using the foam rackas a holder, transfer both the (+)pGLO and (-) pGLO tubes intothe water bath, set at 42°C, forexactly50 seconds. Make sure topush the tubes all the way downin the rack so the bottom of thetubes stick out and make contactwith the warm water. When the50 seconds have passed, placeboth tubes back on ice. For thebest transformation results, thechange from the ice (0°C) to42°C and then back to the icemust be rapid. Incubate tubeson ice for 2 min.

LB-Broth

100 µl

IceIce

Water bath

42°C for 50 sec

9. Remove the rack containing thetubes from the ice and place onthe bench top. Open a tube and,using a new sterile pipet, add250 µl of LB nutrient broth to thetube and reclose it. Repeat witha new sterile pipet for the othertube. Incubate the tubes for 10 min at room temperature.

250 µl

L B/am ppGLO

LB/amp/arapGLO

LB/amppGLO

L B

pGLO

L B

- pG

O

LLB/am p

- pG

O

LLB/am p

+pG

O

LLB/amp/ara

+pG

O

L

+p

GL

O

-pG

LO

QU

ICK

GU

IDE

19

10. Gently flick the closed tubes withyour finger to mix. Using a newsterile pipet for each tube,pipet 100 µl from each of thetubes to the correspondingplates, as shown on the diagramonto the appropriate plates.

11. Use a new sterile loop for eachplate. Spread the suspensionsevenly around the surface of theagar by quickly skating the flatsurface of a new sterile loopback and forth across the platesurface.

12. Stack up your plates and tapethem together. Put your groupname and class period on the bottom of the stack and place thestack upside down in the 37°Cincubator until the next day.

7. While the tubes are sitting onice, label your four agar plateson the bottom (not the lid) as shown on the diagram.

8. Heat shock. Using the foam rackas a holder, transfer both the (+)pGLO and (-) pGLO tubes intothe water bath, set at 42°C, forexactly50 seconds. Make sure topush the tubes all the way downin the rack so the bottom of thetubes stick out and make contactwith the warm water. When the50 seconds have passed, placeboth tubes back on ice. For thebest transformation results, thechange from the ice (0°C) to42°C and then back to the icemust be rapid. Incubate tubeson ice for 2 min.

LB-Broth

100 µl

IceIce

Water bath

42°C for 50 sec

9. Remove the rack containing thetubes from the ice and place onthe bench top. Open a tube and,using a new sterile pipet, add250 µl of LB nutrient broth to thetube and reclose it. Repeat witha new sterile pipet for the othertube. Incubate the tubes for 10 min at room temperature.

250 µl

L B/am ppGLO

LB/amp/arapGLO

LB/amppGLO

L B

pGLO

L B

- pG

O

LLB/am p

- pG

O

LLB/am p

+pG

O

LLB/amp/ara

+pG

O

L

+p

GL

O

-pG

LO

QU

ICK

GU

IDE

19

10. Gently flick the closed tubes withyour finger to mix. Using a newsterile pipet for each tube,pipet 100 µl from each of thetubes to the correspondingplates, as shown on the diagramonto the appropriate plates.

11. Use a new sterile loop for eachplate. Spread the suspensionsevenly around the surface of theagar by quickly skating the flatsurface of a new sterile loopback and forth across the platesurface.

12. Stack up your plates and tapethem together. Put your groupname and class period on the bottom of the stack and place thestack upside down in the 37°Cincubator until the next day.

7. While the tubes are sitting onice, label your four agar plateson the bottom (not the lid) as shown on the diagram.

8. Heat shock. Using the foam rackas a holder, transfer both the (+)pGLO and (-) pGLO tubes intothe water bath, set at 42°C, forexactly50 seconds. Make sure topush the tubes all the way downin the rack so the bottom of thetubes stick out and make contactwith the warm water. When the50 seconds have passed, placeboth tubes back on ice. For thebest transformation results, thechange from the ice (0°C) to42°C and then back to the icemust be rapid. Incubate tubeson ice for 2 min.

LB-Broth

100 µl

IceIce

Water bath

42°C for 50 sec

9. Remove the rack containing thetubes from the ice and place onthe bench top. Open a tube and,using a new sterile pipet, add250 µl of LB nutrient broth to thetube and reclose it. Repeat witha new sterile pipet for the othertube. Incubate the tubes for 10 min at room temperature.

250 µl

L B/am ppGLO

LB/amp/arapGLO

LB/amppGLO

L B

pGLO

L B

- pGO

LLB/am p

- pGO

LLB/am p

+pG

O

LLB/amp/ara

+pGO

L

+p

GL

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GFPII:Transformation-6

IGVPracticeManystudentsfinditchallengingtousetheIntegrativeGenomeViewer(IGV)ontheSPOC.Inthispartofthelab,yourTAwillhelpyoutolearnhowtousetheIGVbyworkingthroughsomeoftheSPOCIGVproblems.