Multiple recombinant plasmids in transformed E Coli? - (Feb/12/2008 )
Hi,
When I do a colony PCR on my transformation plate, sometimes I see colonies that appear to carry 2-3 different recombinant plasmids, each containing 1, 2 or 3 copies of the insert (PCR product).
The PCR product was end-repaired (blunt and phosphorylated) and ligated to a blunt-cut vector, purified and electrotransformed into TOP10 cells.
Any idea why I am not seeing just ONE kind of recombinant plasmid in the colony PCR?
Tim
-timpanister-
Maybe the colonies you're picking are too close together and you're picking more than one at a time.
Try re-streaking some of them and then picking isolated clones.
-Ambrósio-
QUOTE (timpanister @ Feb 12 2008, 01:17 AM)
Hi,
When I do a colony PCR on my transformation plate, sometimes I see colonies that appear to carry 2-3 different recombinant plasmids, each containing 1, 2 or 3 copies of the insert (PCR product).
The PCR product was end-repaired (blunt and phosphorylated) and ligated to a blunt-cut vector, purified and electrotransformed into TOP10 cells.
Any idea why I am not seeing just ONE kind of recombinant plasmid in the colony PCR?
Tim
When I do a colony PCR on my transformation plate, sometimes I see colonies that appear to carry 2-3 different recombinant plasmids, each containing 1, 2 or 3 copies of the insert (PCR product).
The PCR product was end-repaired (blunt and phosphorylated) and ligated to a blunt-cut vector, purified and electrotransformed into TOP10 cells.
Any idea why I am not seeing just ONE kind of recombinant plasmid in the colony PCR?
Tim
Maybe the pcr product come from your ligation mixture.
-WHR-