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Page 1: SiteDirected Mutagenesis (Stratagene protocol) · SiteDirected Mutagenesis (Stratagene protocol) This is the protocol for site‐directed mutagenesis based on the

Site­DirectedMutagenesis(Stratageneprotocol)Thisistheprotocolforsite‐directedmutagenesisbasedontheStratagenekit.Materials:*Pfuturbo*10XPfuturbobuffer*dNTPs(10mM)*Forwardandreverseprimers(0.1ug/uL,seemethodssectionfordesigntips)*dH2O*Dpn1*competentcellsMethods:PrimerDesign*Forwardprimershouldbebetween25and45basesinlengthandcontainthedesiredmutationinthecenterwithcorrectsequencesonbothsides;thereverseprimeristhereversecomplementofthis*PrimersshouldhaveaminimumGCcontentof40%andterminateinoneormoreC'sorG's*Tmshouldbegreaterorequalto78degreesCelsiusandcanbecalculatedasfollows:oTm=81.5+0.41(%GC)‐675/(lengthinbases)‐%mismatchReactionSetupasfollows:Components AmountTemplateDNA(50ng/uL) 1uL10XBuffer 5uLForwardPrimer(0.1ug/uL) 1uLReversePrimer(0.1ug/uL) 1uLdNTPs(10mM) 1uLPfuturbo 1uLdH2O40uLPCRProgramDependingonthelengthoftheplasmid,thisprogramcanbecomeverylong,soitmaybebesttorunovernight.

Page 2: SiteDirected Mutagenesis (Stratagene protocol) · SiteDirected Mutagenesis (Stratagene protocol) This is the protocol for site‐directed mutagenesis based on the

1.95degreesfor1minute2.95degreesfor50seconds,60degreesfor50seconds,68degreesfor1minute/kbofplasmidlength‐‐repeatthisstep17times,or18cyclestotal3.68degreesfor7minutes4.4degreeholdFollowingPCRAdd1uLofDpn1toPCRreaction.Incubateat37degreesCelsiusfor1‐2hourstodigestparentalDNA.Run5uLofthedigestedreactiononagelandcomparetotheundigestedparentalplasmid‐thereshouldbesomedifferenceinbandpattern.Transformintocompetentcells.


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