PCR from viral plaques?

[Peter William Krug]

Alexandra Martins 417 (amartins at pen.gulbenkian.pt) wrote:

: Dear netters,

: Is there a simple protocol for selecting vaccinia virus recombinats
: by doing PCR directly from the lysate of isolated viral plaques (instead
: of dot-blot hybridization)?
: Thanks in advance,

Hi. We use this method for screening HSV plaques for recombinants. 
Basically, we pick the plaques into 1 ml of DMEM w/1% NBS (No skim milk) 
and boil half of it for 10 minutes. We then do two Phenol extractions and
 etOH ppt them. At least with HSV, I have to go up to 35 
cycles to see PCR products on a gel if i PCR from a picked plaque.
In my experience, it is more efficient to grow up a 24-well's
worth of each plaque stock first, then take 30-40% of the well and put
that through the above protocol. 30 cycles works well using this method.

Hope this helps,

Peter Krug

pkrug at bimcore.emory.edu