optimizing CIP

tronni at ktl.fi tronni at ktl.fi
Mon Jan 31 11:03:44 EST 1994


In article <genecutl-300194132312 at kos2mac17.berkeley.edu>, genecutl at mendel.berkeley.edu (gc) writes:
> I CIP and I CIP and I CIP and I still get an unacceptibly high background
> in my ligations.  Does anybody have suggestions for optimizing the CIP
> reaction, or should I just drop out of grad school now?
> 
> -- 
> --gc

Read the protocol of Sambrook, Fritsch & Maniatis, II edition, pages
1.60 - 1.61. I got the same problem as you, but this protocol helped
a lot. The main thing is not to use too much CIP as the carryover
will eat 5'´phosphate groups of your insert in ligation reaction!
It is important to digest CIP completely with proteinase K and
phenol-chloroform extract. After this do the gel purification step.
I always linearize and CIP about 10 - 20 micrograms of vector,
so if the batch proves to work then I have good stuff in fridge
for many years to come.

Cheers,

Tapani Ronni
National Public Health Institute
Mannerheimintie 166
00300 Helsinki
FINLAND




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