noncomplementary primers amd pfu polymerase

[Frank O. Fackelmayer]

Peter Cherepanov wrote:
> 
> Pfu IS less efficient then Taq; before I always tried to use Pfu to avoid
> mutations, and it did work often, but not always.
> I think, the problem is not with your 5' tails - anyway try this reaction
> with Taq (I always do it in parallel).
> After few failures with Pfu, I switched to Pwo polymerase in all my cloning
> work; it is more processive then Pfu, always works better 

You know that Pfu and Pwo are identical in sequence, don´t you?


(makes a bit more
> mistakes though, but if you do not cycle the reaction too many times and
> take care to sequence your construct at the end, it is not going to be a
> problem).

> However, be very carefull with very long primers: if your tails are too long
> PCR might not work at all or be very unspecific. 

no

>The longer the primer the
> more dirty it is. 

no.

>If I need to add a long tail to my PCR fragment (like an
> epitope tag) I split it in two parts which are added in two consequitive
> PCRs, and this works always.
> Also: Pfu, unlike Taq, never works for colony PCR - you need to have clean
> DNA (I think, Pwo does not work too).

true. Pfu is not good for too dirty samples. 


Frank