optimizing CIP

Steve Rodems smrodems at students.wisc.edu
Sun Jan 30 23:14:01 EST 1994


In article <genecutl-300194132312 at kos2mac17.berkeley.edu>,
genecutl at mendel.berkeley.edu (gc) wrote:

> 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

CIP in my hands is highly variable.  One day it works great and one day it
down right sucks.  Mind you I don't pay too much attention to the reaction
conditions.  What I mean is that I cut my vector in whatever REact buffet I
need and then CIP treat in about 100 µl.  I use 10µl of 10x CIP bring it up
to 100µl and then add 3-5µl of CIP (undiluted!) at t=0 and 30'.  Yes, this
is overkill by quite alot, I think companies recomend diluting their CIP
10-fold and using 1 µl but who's got the time!  Just don't tell your boss,
mine gives me shit all the time about wasting enzyme, but then maybe he
should do the minipreps.  Anyway, if you use enough insert it's usually not
a problem to get your clone.  Sometimes, I'll do a control where I ligate
CIP treated vector, transform, and plate it out and I'll get just as many
colonies as if I added insert.  When I miniprep my insert containing
lig/transformations I get 18-18 correct clones.  I don't do this anymore
and most the time I only do about 6-8 minipreps (all you need is one
right?).  I find the less I worry about things the better they go.  Zen
science for me, just gotta believe its gonna work and it will!

Good luck and don't worry, there's more to life than the lab!

Steve



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