In article <Pine.ULT.3.91.961016102221.18991M-100000 at rowan>, SARAH LOUISE
RAWLINGS <slr2 at coventry.ac.uk> wrote:
# I am a student carrying out some reaserch into a protien phosphatase. I
# am trying to insert my DNA which has been blunted with Klenow into a
# vector that hes been cipped with Calf intestinal Phophatase. Upon trying
# to religate the DNA with my vector they do not ligate very easily (if at
# all)...I was wondering if anyone had any ideas on what the problem could
# be.This procedure has been repeated many times with the same lack of
# results.
In the lab I work in, people are very paranoid about CIP, whether it is
contamination with nuclease activity (usually exo), or inability to get rid
of all of it resulting in phosphatasing of the insert by mistake.
Cloning problems I've had have been solved by very careful use of CIP,
including:
1) treatment with CIP for only 15 minutes at 37 degrees, followed by
heat kill for 30 minutes at 65 degrees (which is NOT fully effective)
2) gel purification of the CIP'd vector. If you are going to gel purify
the insert on the same gel as the vector, try to load the CIP-treated
vector LAST and in a lane far away from the insert.
I've not found it particularly helpful to organically extract the CIP'd
vector, probably because I don't manage to get rid of all the phenol...
You might try that, too, though.
I repeat, this might just all be silly superstition with CIP, but cloning
that didn't work in multiple attempts started to work with more careful
CIP use.
Good luck,
Joe
--
- Joe Chou (jchou at cgl.ucsf.edu)
http://devbio-mac1.ucsf.edu/
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