TA cloning : clone contamination

Eric C. Anderson anderson at pharmdec.wustl.edu
Sat Apr 1 20:44:14 EST 1995


In article <3lebf8$ntf at yama.mcc.ac.uk>, @fs1.ho.man.ac.uk (chukins) wrote:

> 
> I cloned a PCR product using TA cloning, I picked white colonies , 
> analysed rapidly with PCR using the same primers I originally used for my 
> PCR product , they were all positive.
> After O/N culture in liquid media, I did  streak my white colonies, 
> surprisingly I found blue colonies growing on my plates that had X-Gal.
> Is there any logical explanation for this apart from the very remote 
> possibility of contamination?Please help.

how long did you let the clones grow on the original plate?  i've had
colonies that looked white and then the next day started to turn blue. 
also...how large is your insert?  in the Invitrogen TA Vector (the only one
i have experience with, no commercial involvement/interest here) small
inserts (<500bp) may not sufficiently interrupt the lacZ gene and your
transformants may be light blue in color rather than white.

of course, it could just be contamination but this is a thought anyway. 
have you screened the colonies that grew up as blue on the second plating
screen?  if not i suggest that you do and see what happens.

eric
 
-- 
eric c. anderson                                
anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)862-2435 (home)
660 s. euclid box 8103                           (314)362-7058 (FAX)
st. louis, mo 63110



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