An Economic PCR Enhancer for GCRich PCR Templates

SincePCRisoftenproblematiconGC-richtemplates,wehaveevaluateddifferentPCRenhancingadditivesandgeneratedanCombinatorialEnhancerSolution(CES).Wetestedthissolutiononapprox.50differentprimerpairsusingseveraldifferentpolymerases

The5xconcentratedCombinatorialEnhancerSolution(CES)contains:

2.7Mbetaine6.7mMDTT6.7%DMSOand55μg/mlBSA

AddthissolutiontoyourPCRandseeifitworks

SomehintsifyourPCRstilldoesn""twork.

1. Increasetheannealing-temperatureincomparisontoPCRwithoutCES
2. TrydifferentMgconcentrations
3. Checkifyoudidn""taddtomuchtemplate(morethan20ng/PCRcanalreadyinhibitthereaction!)
4. TitratetheCES.Insomecases,thiscanmakeabigdifference

WetestedthisenhanceralsoonPCRwhichworksfinewithoutadditiveandwevirtuallyobservednonegativeeffects.Therefore,wearenowaddingthisenhancerforallourroutinePCR.

Notes

1. Betaine:Wepreparea5Mstocksolution,whichdissolvesfinewhenautoclaving.
2. OurclassicPCRreactionsareperformedinthefollowingbuffer(finalconcentration)optimizedforTaqDNAPolymerase(SupplementationwithPfucanincreasetheproductqualityandyield,notonlyenhancingtheproof-reADIng!)

65mMTris–HCl16.6mM(NH4)₂SO4,3.1mMMgCl₂0.01%(v/v)Tween20pH8.8(or8.0)andsupplementedwith5-10pico-molesofeacholigo(perreaction)and0.5μlof25mMdNTPmix.

Ifthisenhancerhelpsyoutoimproveyourexperimentsandhelpsyoutosavetimeandmoney,befairandcitethework:

M.Ralser,R.Querfurth,H.J.Warnatz,H.Lehrach,M.L.YaspoandS.KrobitschAnefficientandeconomicenhancermixforPCR.BiochemBiophysResCommun.2006Sep1;347(3):747-51.Epub2006Jul5