Expand PCR and TA cloning kits?

Margon Vandongen vando006 at mc.duke.edu
Thu Apr 4 09:50:47 EST 1996


John,

To your question: no, you do not have to purify your nukes away after
this as long as you do not use more than one (1) ul in your TA ligation.
Just leave everything in there and it will work fine. As to PFU versus
PWO: I have never tried to clone a fragment in TA after PCR with a 
mixture of PWO and Taq (= Taqextender). An E-mail message from a BM 
salesrep told me that adding A's was not necessary, he claimed greater
than 80% A overhangs. I can tell you that this is not the case with PFU
so maybe these enzymes are different although I would not exactly know
how.(They are both proofreading).
There is a nice reference on using mixtures of pcr enzymes which was
one of the reasons we started trying this: Wayne M Barnes "PCR 
amplification of up to 35 kb DNA with high fidelity and high yield
from bacteriophage templates", PNAS vol 91 2216-2220 March 1994.
There was a little follow up reply in Biotechniques in which he actually
states the ratio's used of the different enzymes: 1/16 PFU or 1/50
Deep vent. I do not have the full reference, only the pagenumber but that 
is not helping much I'm afraid.
We do not use the method to PCR long templates but we noticed that with 
some of our difficult PCR's (GC rich regions etc) we got a much better
product. Hope this helps,

Margon. 




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