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
