A GOOD KIT FOR PCR CLONING

Daniela Sciaky reedc at iac.net
Tue Jan 31 20:58:20 EST 1995


In article <drm21-3001951307000001 at drm-mac1.welc.cam.ac.uk>,
drm21 at mole.bio.cam.ac.uk (David Micklem) wrote:

> In article <reedc-2901952101470001 at ip043167.iac.net>, reedc at iac.net (Reed
> R. Corderman) wrote:
> 
> > In article <Pine.ULT.3.91a.950126165357.24016A-100000 at kepler.unh.edu>,
> > xz at KEPLER.UNH.EDU (Xin-Hua Zhou) wrote:
> > 
> > >  I am looking for a good kit for PCR cloning. Can you give me some ideas 
> > > , based on your EXPERENCE?

> Er.  Maybe I'm missing something here.  Don't you need a ligation in there
> somewhere between the PCR and the transformation?  If not, I'd be very
> interested to hear the protocol!
> 
Yes. I stand corrected. Complete protocol:
1 microliter 10x ligation buffer (I use NEB ligase and their 10x)
1 microliter pCRII vector provided in kit
3 microliters PCR
1 microliter ligase (NEB)
4 microliters water
   proceed as directed in kit but plate all the cells.
After taking about 2-3 months to blunt end ligate via normal routes, I
used the TA system and in one week got the clone and the sequence. Yes!!!!

PS. If you do RT PCR and have a lot of oligos left in the reaction you
need to purify the PCR product first. We use Centricon or similar. Gel
purification just fails. The ends of the PCR product are fragile???



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