szcooley at chip.ucdavis.edu
Thu Oct 17 07:01:59 EST 1996
SARAH LOUISE RAWLINGS (slr2 at coventry.ac.uk) wrote:
: I was wondering if anybody could help me with my molecular bioligy
: 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
: The procedure being followed is that in Maniati's and the chemicals being
: used are not at fault. Thanks.
: Sarah Louise Rawlings
: Bsc Biochemistry (coventry University)
: SLR2 at coventry.ac uk
A number of things could be happening:
1) Did you know that CIP is a very sticky protein. It is ver hard to get
rid of after the CIP reaction and binds to the ends of the molecule, which
will prevent ligation. The enzyme contains zinc so I treat the reaction
with 1/10th vol. 200 mM EGTA. Note: thats EGTA not EDTA. Heat to 65 oC for
1 hr. and then extract with phenol/chloroform and precipitate.
2) Though Klenow is the obvious enzyme for blunting, I use T4 polymerase.
Its very effective provided you are careful to run the reaction at 12 oC
to limit the 3' to 5' exo activity.
3) additionally it would be helpful to know if you are getting any
colonies and if so are there any white ones and if so is there no insert
or the wrong insert. All of this tells you something of the original
starting materials, DNA and cells
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