xho1 ligation problem

Peter Ianakiev ianakiev at genome.wi.mit.edu
Fri Jan 25 10:40:55 EST 2002


Hi Amy,
There is few controls for transformation that might help you to find out what
is going on:
1.Vector  cut and purified.
2. Vector + ligase
3. Vector + Ligase + Kinase

First control will give you the amount of uncut vector, second - the
dephosphorilation, and the third will give you information about the ends of
the vector and efficiency of purification from CIP.
Hope this will work.
Also it seems that you have wrong ratio Vector:Insert. It should be close to
1:1 by means of ends.
Regards
Peter

Amy Prasch wrote:

> 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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