xho1 ligation problem

Amy Prasch alprasch at students.wisc.edu
Fri Dec 14 17:58:34 EST 2001

I am trying to make a construct and am stuck on this step. I want to cut my
insert out of the donor vector using xho1, linearize my acceptor vector
using xho1 ( and CIAP treat) and then ligate the xho1 fragment into the
acceptor vector. The xho1 appears to be cutting both the vector and insert
well. However, after the ligation and transformation I get no colonies.
When I look at my ligation reaction on the gel I see the vector band, a
faint insert band and then a ladder in between them which to me looks like
my insert ligating to each other in multiple copies. My interpretation of
this is that my insert pieces can ligate to each other but not to the
vector. Does anyone have any ideas about what might be going on here or how
to solve the problem?
Any help is greatly appreciated.


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