Megaprimer PCR

[Roland Hübner]

In article <366FB62C.13C30B31 at liv.ac.uk>, "Richard V. Giles"
<giles at liv.ac.uk> wrote:

>  Csaba Jeney wrote:
>  > 
>  > We are trying to get a PCR product using a very long primer pair (60bp
>  > each, 20 bp anealing length) but until now there is nothing to glad.
>  > We already have changed the annealing temperature, the Mg
>  > concentration and the next step will be the primer concentration.
>  > Could anybody out there suggest other things and/or suggest the ranges
>  > of these variables to get of rid this problem.
>  > 
>  > Thank you in advance.
>  > 
>  > Csaba
>  
>  My experience with such primers (I use them to introduce phage RNA pol
>  sites) is that they behave just like shorter primers (missing the pol
>  sequences).

 Caution: in case you use a "proofreading" pol or a mix where it is present
at a high rate, your long primer can get nibbled if not protected...

All the best,
 Roland