TOPOTACloning

InstructionsModifiedforSimpsonLab

1.PCR:PerformastandardPCRreactionusingtaqpolymerase.(DonotusetflasthePCRproductmusthavea3"adenineoverhangthatonlytaqgenerates.)Includean72°extensionstepfor7to30minutesattheendofthereactiontoensurethatallproductsarefulllengthand3"adenylated.Checkresultsonagelandproceedimmediatelytothecloningreaction.

2.PreparefortheTransformationReaction.

a.Setwaterbathto42°C.

b.WarmSOCmedium(Box2)toroomtemperature.

c.Warmpre-pouredLBplateswith50µg/mlampicillinat37°for30minutes.

d.Spread40µlof40mg/mlX-gal(infoil-coatedboxin-20°Cfreezer)oneachLBplateandincubateat37°untilreadyforuse.

e.Thaw1vialofOneShotcellsforeverythreetransformations.

3.SetupCloningReaction:Wedoourreactionsat1/3themanufacturer"srecommendedvolumes,andsouseonevialofOneShotcellsforeverythreetransformations.Thereactionmixtureisasfollows:

FreshPCRproduct1.33µl(fromStep1)

SaltSolution0.33µl(inkitin-20°Cfreezer)

TOPOVector0.33µl(inkitin-20°Cfreezer)

FINALVOLUME2.00µl

4.Incubate:Gentlymix(donotuseapipettor!)thereactionsandincubateatroomtemperaturefor5minutes.ForlargePCRproducts(>1kb)orforapoolofPCRproducts,youcanincreasethetimeupto30minutes.Afterincubation,placethereactiononice(orstoreovernightat-20°C).

5.TransformintoCompetentCells:

a.Add16µloftheOneShotChemicallyCompetentE.colicellstoeachcloningreaction.EachvialofOneShotwillyieldenoughforthreecloningreactions.

b.Incubateonicefor5to30minutes.

c.Heat-shockthecellsfor30secondsat42°Cwithoutshaking.

d.Immediatetransferthetubestoice.

e.Add83µloftheroom-temperatureSOCmedium.

f.Incubatefor1hourinthe37°shakingincubatorat200rpm.

g.Usingsteriletechnique,spread40µlfromeachtransformationontoaprewarmedLBplate.Letstandright-sideupforabout5minutestoallowthecellstoadheretotheagar,theninvertandincubateovernightat37°C.

6.SelectandAnalyzeColonies:Selectandanalyzeonlywhiteorlightbluecolonies;darkbluecolonieshavenotreceivedinserts.WeanalyzecoloniesviaPCR.Therearetwooptionsfordoingthiswhicharelistedbelow.Sample15-20coloniesandmarkeachwithanumberontheundersideoftheplateforreferencepurposes.

Option1:YoucanPCRdirectlyfromacolony.SimplysetupastandardPCRreaction,thentakeaplugfromeachcolonytobesampledusingaP10pipettipandapipettorsetto10µl.YouwillhavetopipetupanddownonceortwicetogetthecellstodetachfromthepipettipandintoyourPCRreaction.Thishastheadvantageofbeingquickandeasy,butthecoloniesarefrequentlysomessedupbythesamplingthattheyarehardtoresampleshouldyouneedtodothis.

Option2:Growyourcoloniesinmicrofugetubesin200µl(orless?)LBbrothwith50µg/mlampicillinat37°intheshakingincubatorfor1or2days.E.coliclumpscanbedispersedbyvortexingforaminuteortwo.CellsUSPensionsshouldbecloudywhenvortexed.OneµlservesastemplateforPCRreactions.Thistakesmoretimethanthefirstoption,buthastheadvantageofallowingformultiplePCRreactionsfromthesamecolony.

Notallofthewhitecolonieswillhavebeentransformedwithfull-lengthPCRproduct(theymayhavebeentransformedwithprimerorpartialproduct),sonotallwillyieldPCRproductfromStep6.(Thisiswhyyousampledmorethan10colonies.)Ofthosethatdidamplifysuccessfully,standardoperatingprocedureistosequence10,althoughthiscanbevarieddependingonthepurposeofthestudyandbudgetaryconstraints.(Oneoptionistosequenceoneprimeronlyfor10coloniestoassessvariation,thentosequencethereverseprimeronlyforonecolonyrepresentingeachuniquesequencetype.)