xho1 ligation problem

AvV avvliet at ision.nl
Fri Dec 14 19:21:36 EST 2001


- Leave out the CIAP, and try the ligation again. This is the most likely
option, as CIAP is a sh%t enzyme. Still no colonies?
- (Re)ligate only vector and check. Still no colonies?
- check competent cells using plasmid controls
- check ligase, ATP

Hope this helps
Arnoud

--

"Amy Prasch" <alprasch at students.wisc.edu> wrote in message
news:4.1.20011214164526.009d27a0 at students.wisc.edu...
> Hello,
> 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.
> Thanks,
> Amy
>
> ---





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