Direct PCR from E. coli colonies

Pierre-Alain Menoud menoud at cmu.unige.ch
Mon Jul 10 02:52:36 EST 1995


In article <pbv.65 at rs.uovs.ac.za>,
   pbv at rs.uovs.ac.za (B VISSER * x2818 Plantkunde *) wrote:
>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.
There is a protocol which worked every time so far. I use it every time it is 
possible:

1) Make up the following reaction mix in bulk for n+1 reaction:

per reaction:	water 			14.9 	ul
		10XPCR buffer		2 	ul
		5 mM dNTPs		1 	ul
		primer 1(10 pmol/ul)	1	ul
		Primer 2(10 pmol/ul)	1	ul
		taq polymerase		0.1	ul

TOTAL					20 ul

Aliquot 20 ul into each tube.
Gently stab the center of a well-isolated plaque with a new toothpick, and 
swirl the toothpick briefly in the PCR reaction mix. Then place the toothpick 
into an appropriate culture tube (filled with culture medium) or streak the 
remaining of the colony onto an agar plate. Repeat for each plaque.
Cover each reaction with paraffin oil as a single drop per tube and amplify. 
Usually 94oC, 2 min.
	(94oC, 1 min., 55oC, 1 min., 72oC, 1 min.) x 25 cycles
	72oC 5 min.
I load all the PCR reaction into an agarose gel.

Protocol from "General application of PCR to gene cloning and manipulation". 
T. Clackson, D. Guessow, T. Jones. Chapter 11, pp.:210-212. I don't remember 
the book title, but it might be "PCR, A practical Approach" or so.

Hope it will help

Pierre-Alain
menoud at cmu.unige.ch 



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