TransformingCompetentCellswithPlasmidDNA

Reference:
ProtocolprovidedbyPGCScientificsCorporation.
Summary:
Transformationtheheritablemodificationofthepropertiesofacompetentbacteriumby
DNAfromanotherbacterialstrain.
DH5competentcellswillbeused(e.g.,PGCScientific).AplasmidwithDNA
resistanttoampicillinfromanotherbacterialstrain(foreignDNA)willbeintroducedto
theDNAofthecompetentbacterium(hostDNA)andstreakedonLBagarplateswith
antibiotic.CellswiththeforeignDNAwillberesistanttoampicillin,resultingincolonies
onLBagarwithampicillin.CellswithouttheforeignDNAwilllyse!!
Procedure:
1. Removecellsfrom70oCandletthawonice.
WARNING:Whenhandlingfrozencompetentcells,itisimportanttoKEEPIT
COOL!Frozencellsareverysensitivetobeingwarmandwillnotworkaswellifnot
keptoniceasmuchaspossible.
2. Placethecellsonbenchtoptothaw.Replaceonice.
3. Aftermixinggently,aliquot~25Lofcellsintochilled,17x100mmpolypropylene
tube(s),e.g.,Falcon2059.Note:25LisenoughforTransformationwhile50Lis
requiredforLigation.
4. Add1.25LorlessplasmidDNA(thevolumeofDNAsolutionshouldnotexceed
5%ofthevolumeofthecompetentcellsornomorethan50nginavolumeof10L
orless).Youdontneedverymuchplasmidtogettransformedcolonies.Generally
around20ng(nanograms)isenough.
5. Gentlyswirltube(s)forafewsecondstomixbyfingerflickingtube.Note:Ifa
controlisdesired,repeatthisstepwith12L(1020ng)ofpUC19inaseparate
tube.
6. Thenincubatetubeonicefor30minutes.
7. Heatshocktube(s)byplacingin42oCwaterbathfor~45secondswithoutshaking.
8. Replacetube(s)onicefor25minutes.

9. Add900LofS.O.C.(2%Tryptone,0.5%YeastExtract,0.4%glucose,10mM
NaCl,2.5mMKCl,10mMMgCl2,&MgSO4).
10. Gentlyshakeandincubatetube(s)at~200rpmfor3060minutesat37oC.
11. SpreadonLBagarplatescontainingappropriateantibiotic(e.g.,100g/mL
ampicillinforcontrolpUC19).Tip:Spreadbyevenlyspreadingcellsovertheplate
usingahockeypuckspreader.Besurethatyouhavedippedyourspreaderin
ethanolandflameditbeforespreading.
12. Placeinthe37oCincubatorwiththeagarsideupandthelidsidedown.Incubate
overnightandremovetofridge(4oC)sometimethenextday.
Reagents/Solutions:
1. SOCMedium(1Liter)
800mLofdeionizedH2O
20gBactoTryptone
5gBactoYeastExtract
0.5gNaCl
Shakeuntilthesoluteshavedissolved.Add10mLofa250mMsolutionofKCl.
(Thissolutionismadebydissolving1.86gofKClin100mLofdeionizedH2O.)
adjustpHto7.0with5NNaOH(~0.2mL).Adjustthevolumeofthesolutionto1
LiterwithdeionizedH2O.Autoclave.
Justbeforeuse,add5mLofasterilesolutionof2MMgCl2.(Thissolutionismade
bydissolving19gofMgCl2in90mLofdeionizedH2O.Adjustthevolumeofthe
solutionto100mLwithdeionizedH2O.Autoclave).
*20mMglucose(AfterSOChasbeenautoclaved,allowtocoolto60oCorlessand
thenadd20mLofasterile1Msolutionglucose.Thissolutionismadebydissolving
18gofglucosein90mLofdeionizedH2O.afterthesugarhasdissolved,adjust
volumeofthesolutionto100mLwithdeionizedH2Oandsterilizebyfiltration
througha0.22micronfilter.)
2. 3MNaOAcpH5.2(1Liter)
IfFW=82.03g/mol,add:
246.1gCH3COONa(anhydrous)
800mLdeionizedH2O
pHto5.2withGlacialAceticAcid

Bringtovolumeof1Liter
Autoclave

3. LBagarplates(1Liter)

800mLdH2O
10gBactoTryptone
5gBactoYeastextract
10gNaCl
pHto7.0
Bringtovolumeof1Liter
*Autoclave
*Justbeforeautoclaving,add15gofBactoagarsolidifyingagent/1LiterLB
Medium

UsefulTables:

AntibioticSolutions

Ampicillin
Carbenicillin
Chlor
amphenicol
Kanamycin
Streptomycin
Tetracycline

Stocksolution
concentrations
storage
50mg/ml
inH20
20oC
50mg/ml
inH20
20oC
34mg/ml
inethanol
20oC
10mg/ml
inH20
20oC
10mg/ml
inH20
20oC
50mg/ml

Workingconcentration
Stringent
Relaxed
plasmids
plasmids
20g/mL

60g/mL

20g/mL

60g/mL

25g/mL

170g/mL

10g/mL

50g/mL

10g/mL

50g/mL

inethanol

20oC

10g/mL

50g/mL

StocksolutionsofantibioticsdissolvedinH2Oshouldbesterilizedbyfiltrationthrougha0.22micronfilter.
Antibioticsdissolvedinethanolneednotbesterilized.Storesolutioninlighttightcontainers.
b

Magnesiumionsareantagonistsoftetracycline.Usemediawithoutmagnesiumsalts(e.g.,LBMedium)forselection
ofbacteriaresistanttotetracycline.

MediaContainingAgarorAgarose

Prepareliquidmediaaccordingtotherecipesgivenabove.Justbeforeautoclaving,add
oneofthefollowing:
Bactoagar
(forplates)15g/Liter
Bactoagar
(fortopagar)7g/Liter
Agarose
(forplates)15g/Liter
Agarose
(fortopagar)7g/Liter