Problem cloning PCR product into pCR-Script
egreif at cc.umanitoba.ca
Wed Mar 11 12:23:19 EST 1998
In article <3506ABF0.45FA at nospam.ic.ac.uk>, k.desmet at nospam.ic.ac.uk wrote:
> We routinely use pCR-Script from Stratagene to clone our PCR products.
> After transformation we get around 50% white colonies on IPTG/X-gal
> plates; but we have a similar ratio on a vector-only control plate. When
> we pick colonies for minipreps (from the plate that should have clones
> with our insert), some of the white ones are pCR-Script with our insert,
> but the majority have what looks like vector only with deletions. The
> vector is 2.9 kb, but these clones have single bands ranging in size
> from 1.5 kb to around 2.9 kb.
If you are getting deletions then you must have a contaminating nuclease.
This happens to us quite a bit, usually in the summer beleive it or not.
We simply throw out all the solutions or autoclave the ones you can and
repeat the cloning experiment. This usually works.
dboyd at cc.umanitoba.ca
hey hey hey it was the DNA
hey hey hey that made me this way
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