Restriction sites.

Aawara Chowdhury aawara at FEMA-trailer.org
Wed Jun 28 21:43:48 EST 2006


In <mailman.41.1151546583.20007.methods at net.bio.net>,
 Ezhil Subbian <eksubbs at yahoo.com> wrote:

> Hi,
>
> I designed primers to add new restriction sites but forgot to add any "clamp" sequences at either end. I have a KpnI site at one end and a BamHI site at the other.  I realise my mistake. I'm curious to see if anyone has had any succes with digesting inserts with the restirction site at the very end.  Or if its a lost case and I should just start over with fresh primers.  
>
> Thanks
> E

There are several things you can do to clone with your existing primers:

a) Phosphorylate the primers using T4 polynucleotide kinase.  Then do the
   PCR using an error-correcting thermostable polymerase (Vent/Pfu/Pwo etc.)
   Purify the PCR product, and self-ligate it (37oC for 1 - 2 hours).  This
   ligates your product into dimers/trimers/tetramers etc.  NOW, cut with
   KpnI and BamHI.  Because you're cutting a concatamer, their recognition
   sites are no longer at the ends.  Purify the cut product, and ligate it
   with your cut vector.

b) As an alternative, if you use Taq for PCR, just clone the product into
   any TA-cloning vector.  Do a miniprep, and cut your insert out of your
   new TA-vector.

Good luck!
AC
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