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...
>
>Hi Netters
>
>Just a quick question.  I recently PCR amplified cDNA fragments directly 
>from plaques by first incubating the phage particles at 70oC for 10 
minutes 
>to disrupt the capsid protein.  This liberated the phage DNA which could 
>then be used for the amplification.  The amplified DNA was digested and 
then 
>cloned into a shuttle plasmid vector.
>
>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


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 mailing list