cloning a pfu amplified fragment in T vector

Soenke Behrends via methods%40net.bio.net (by s.behrends from tu-bs.de)
Sat Feb 6 15:00:30 EST 2010


Hi,

I always used Taq for TA cloning after Pfu amplification without prior 
purification. It always worked fine. I would give it a shot and if you 
should have problems you can try what they say in the manual. Maybe they 
are theoretically right, but the Pfu is somewhat less active after all 
the cycles of the PCR reaction and so its presence does not matter anymore?

Kind regards
Soenke

Azam Rahimpour schrieb:
> Hi
> I need to clone a pfu amplified fragment in t-vector, in the vector manual it is said that pfu pcr product must be purified before a tailing by tag because pfu can remove a overhangs by its proofreading activity, but in commercial high fidelity enzyme mixes which include a proofreading enzyme with tag we can use pcr products directly for t/a ligation, so why in the latter case A overhangs are not removed?
>
> regards 
>
>
>
>       
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-- 
Prof. Dr. med. Soenke Behrends
Institut für Pharmakologie, Toxikologie und Klinische Pharmazie
Mendelssohnstr. 1
38106 Braunschweig
Tel.: 0531 391 5604



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