HELP with PCR cloning

nbr at ac.dal.ca nbr at ac.dal.ca
Wed Oct 23 14:16:12 EST 1991


In article <1441 at chem.ucsd.EDU>, hsb at chem.ucsd.edu (Hirdeypal S. Bhathal) writes:
> I tried to subclone RACE PCR products into Hinc II site of pUC-19 after
> blunt ending them with T4 polymerase. On gel, it seems that the ligation worked 
> but I never got a white colony. I am planning to do PCR with primer having
> Eco RI site. I'll appericiate if anyone can give some suggestion.
> Thanks
> 
> Hirdeypal
> hsb at chem.ucsd.edu
>    
Have you looked at the blue colonies?  We found that the blue or light blue ones sometimes carried the cloned PCR fragment, and we no longer do
colour screening.
Now we use PCR to amplify any insert DNA directly from a small bit of cells picked from each colony, followed by electrophoresis in agarose.

Bruce Ramsey
Marine Gene Probe Lab
Dalhousie University
Halifax, Nova Scotia
Canada



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