cloning of pcr product - (Jan/17/2002 )
When ever i ligating the pcr amplified product ~1kb into prset A vector, iam not seeing the recombinants, this is
xho I digested product.please any body suggest me what will be the technical problum in this ligation.
Thanks in advance
Did you add enough bases onto your primer after the XhoI site as many enzymes require more DNA than just the restriction site to cleave efficiently, the back of the NEB catalogue has a good guide for this. I routinely use an extra six bases 5' to site which seems to work most of the time!
Xho I is a difficult enzyme to cut close to the end. Try a PCR cloning vector.