Phosphorylation of Pfu PCR-products

Oliver Sorgenfrei oliver at molgen.biologie.uni-marburg.de
Fri May 9 08:50:55 EST 1997


> Someone stated recently that it is necessary to phosphorylate Pfu PCR
> products prior to e.g. EcoRV subcloning. Is this really necessary?

Sometimes it is necessary. Your PCR product has no 5'phosphate unless
you have used 5'phosphorylated prime (look up you invoive). On the other
hand if you open your vector just by restriction digestion it has
phosphat groups. Thus in principle you can ligate your PCR product
without phosphorylation in your vector.

But be aware that your vector can also religate. If you have a good
possibilty to select for clones with insert (blue/white), it's no big
problem. Otherwise it can be a problem.

Hope that helps.

Oliver



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