kevin.a.morano at uth.tmc.edu
Fri Jun 8 17:04:39 EST 2001
I had the exact same problem while a grad student at Davis (Micro). The
problem was usually that the PCR fragment was poorly cut. Try this:
1) Gel purify PCR product with kit of choice.
2) Phenol/chloroform your kit-purified band twice.
3) EtOH ppt band, wash twice with 70% EtOH.
4) Run a few microliters on gel to make sure you still have it.
5) Cut the rest OVERNIGHT with your restriction enzymes.
6) Gel purify it again, or phenol/chloroform and ppt.
7) Resuspend in water (not TE). Ligate 2 hours on benchtop. Transform.
This is a lot of work, but it really helps. Alternatively, if you can
afford it, buy one of the dA-dT cloning kits, like Promega's, and follow
the instructions. It almost always works, and you are using Taq
polymerase, which is compatible.
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