Direct PCR from E. coli colonies
William D. Woolfolk
wdw7m at virginia.edu
Tue Jul 11 07:37:33 EST 1995
In article <pbv.65 at rs.uovs.ac.za>, pbv at rs.uovs.ac.za says...
>Just a quick question. I recently PCR amplified cDNA fragments directly
>from plaques by first incubating the phage particles at 70oC for 10
>to disrupt the capsid protein. This liberated the phage DNA which could
>then be used for the amplification. The amplified DNA was digested and
>cloned into a shuttle plasmid vector.
>I am now testing whether the digested cDNA was successfully cloned into
>plasmid vector, but instead of minipreps, I want to amplify the inserts
>directly from E. coli colonies. Can the same protocol used for the
>DNA be used directly for amplification of DNA from these colonies? If
>could you suggest a publication where I could find a suitable protocol?
>Thanks in advance.
>pbv at rs.uovs.ac.za
Yes, you can use a similar method. I have frequently amplified directly
from bacteria by pulling up a small amount of bacterial material with a
pipet tip, and adding it to a 50ul PCR reaction with the Taq omitted. I
heat to 95C for 15 minutes,then add Taq and cycle as I would for normal
DNA template-based PCR. The only problem I ever had was using a massive
clump of bacteria, which decreased amplification some. A small swipe is
more than adequate for PCR, so do not overdo it. Good luck.
More information about the Methods