Pfu/Taq in PCR reactions

pgegen at rnaworld.bio.ukans.edu pgegen at rnaworld.bio.ukans.edu
Thu Mar 9 18:49:51 EST 1995


In <Pine.DYN.3.91.950303125117.10091B-100000 at uxa.cso.uiuc.edu>, hodges bradley l <segdoh at uxa.cso.uiuc.edu> writes:
>It is better to have just TAq for TA cloning.  Pfu added to the mix will 
>increase fidelity of replication and also allows the amplification of 
>longer templates.  I don't think Pfu will cleave off terminal A 
>overhangs.  Or if it does the effect on TA cloning may be minimal.  So if 
>you are cloning PCR products less than 1.5Kb, TAq in your mix will be 
>sufficient.
>
WHY is everyone so bent on using TA vectors? My understanding is that cloning a 1-base overhang is less efficient than blunt-end cloning. If you amplify with a mixture of Taq + a proofreading polymerase (Pfu, Pli, etc.), you get blunt-ended product which can be cloned into any vector.

Dr. Peter Gegenheimer
Dept Biochem., University of Kansas




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