Direct PCR from E. coli colonies

lmc at sevax2 lmc at sevax2
Wed Jul 12 05:41:37 EST 1995


In article <pbv.65 at rs.uovs.ac.za>, pbv at rs.uovs.ac.za (B VISSER * x2818 Plantkunde *) writes:
> I am now testing whether the digested cDNA was successfully cloned into the 
> plasmid vector, but instead of minipreps, I want to amplify the inserts 
> directly from E. coli colonies.  Can the same protocol used for the phage 
> DNA be used directly for amplification of DNA from these colonies?  If not, 
> could you suggest a publication where I could find a suitable protocol?  
> Thanks in advance.
> 
> Botma Visser
> pbv at rs.uovs.ac.za

You just have to pick a colony with a sterile toothpick into a 10 ul PCR
reaction and then to an LB+amp plate to keep the strain. Then do a routine
PCR. I have used it hundreds of times to screen for recombinant plasmids
before doing minipreps. It should work just as well with larger volumes.
A protocol including  cycle-sequencing is described in:
Rosenthal et al. Nucleic Acids Res. 21, 173-174 (1993)

Good luck

Luis M. Corrochano
Departamento de Genetica
Universidad de Sevilla
Spain
LMC at cica.es




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