xho1 ligation problem

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sat Dec 15 03:24:13 EST 2001


Dear Amy,

> 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) 

you might use SAP instead (inactivated bt heat)

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?

in such a case, try to include all controls possible in your experiment 
(vector alone, insert alone, try vector + insert at different reatios, even 
control reactions for the ligation and transformation steps could be necessary 
to locate where exactly you get stuck. 
I assume that antibiotics and resitstance genes match?

Regards, 
Wo
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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.o.C.

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