Direct PCR from E. coli colonies (GENOMIC???)
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!)
> Luis M. Corrochano
> Departamento de Genetica
> Universidad de Sevilla
> LMC at cica.es
More information about the Methods