Help!!!! how to clone a PCR fragment?
Dr. Colin Rasmussen
colin_rasmussen at darwin.biochem.ualberta.ca
Thu Aug 3 19:54:14 EST 1995
<Pine.AUX.3.91.950803180003.17417A-100000 at mail.med.cornell.edu>,
hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) wrote:
> I have a cloning project that involves inserting a PCR fragment with one
> cohesive (Bam HI) and one blunt end into pBS.
We usually do the following. After PCR we EtOH precipitate the DNA. We
then resuspend in Klenow reaction buffer along with dNTPs, 1 mM ATP and
add Klenow and T4 PNK. We then run out on an LMP agarose gel, excise the
band and clone into pGEM cut with SmaI and CIPd. So far it has never
failed. One other you could try to obviate the need for the Klenow is to
use Pfu which leaves a blunt-end.
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