TA Clonning

David Micklem dmicklem at cmgm.nospam.invalid
Mon Sep 10 11:03:36 EST 2001


In article <9nghkc$146e$1 at news.doit.wisc.edu>, Peter Guo
<yuyuanguo at students.wisc.edu> wrote:

>Hi, friends:
>
>I tried to clone a PCR product. However, after transform the Top10
>compentence cells, I did not get clones. The PCR is good. I phosphoralted
>the 5' end of the primer. I used Invitragen Company's kits. Ligation
>condition is at 16 degree centigrade. I think it should be at 4 degree
>centigrade. Is this correct?
>
>Would somebody tell me how to treat the problem.
>

Hi Peter,

For you specific question: no 4C is not necessary. I always do all
ligations at room temperature and that works fine.

Did positive and negative control transformations work OK?

Which Invitrogen kit did you use? I think phosporylation is needed for
TA-cloning kits, but a bad thing for the (fantastic, in my experience)
TOPO-TA kits.

Did you gel purify your PCR product? If so, did you make sure to cut
out the band on a long-wave UV gel box and to minimise exposure to the
UV? In my experience, exposure of DNA to short wave UV is by far the 
most common reason for getting no colonies after transformation
assuming the competent cells are alive.

HTH,

David

-- 
D.R. Micklem,              Time flies like an arrow... 
Dept. of Anatomy,          Fruit flies like a banana.
Cambridge University,      Email:dmicklem at cmgm.stanford.edu 
Cambridge, UK              Phone: +44 (1223) 333776
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