Direct PCR from E. coli colonies (GENOMIC???)

Richard_Heath heath at mbcf.stjude.org
Thu Jul 13 12:47:06 EST 1995


In article <1995Jul12.114137 at sevax2>, lmc at sevax2 writes:
> 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

Has anybody used this successfuly for amplifying a gene from *genomic* 
E. coli DNA?  If so, what kind of yield did you get from how many cycles?
(I'm thinking of trying it, but just want a few pointers first!)

TIA

Richard 

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



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