ligation problem

Kevin Morano 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.

Kevin




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