LambdaGem-11 cloning problems
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Jun 30 11:52:36 EST 1999
In article <3779F2A6.60DA1A1E at u.arizona.edu>, Susanne Rafelski
<susanner at u.arizona.edu> writes
>Self ligation of the insert was also checked on FIGE, and showed that
>least some of the insert can ligate at both ends
That looks like the problem. Partially digested DNA should all ligate if
the sites are intact i.e. no phosphatase or nuclease has got near them.
What happens if you heat kill the Mbo I RE digestion, EtOH ppt,
resuspend and try to ligate that? If it will ligate better than the gel
purified DNA then I would look at the elution as the problem. Can you
also ligate Mbo I digested lambda (dam-). If that ligates OK then your
RE is OK etc.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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