Making your own TOPO vector - (Nov/10/2006 )
Hi,
I was wondering if anyone in here have tried to make their own TOPO vector, eg. making pGL3 availible for the TOPO kloning strategy.?
If so, would you like to share your knowledge, I would really like to be able to do this since the Invitrogen costs for such an adaption is really large!
Best,
Mads Hollegaard
-hollegaard-
if memory serves OpenWetWare has a cloning strategy to make a plasmid which contains AhdI sites, that when cleaved produces 3' T overhangs.
another way would be to cut your plasmid with an blunt end enzyme, then add a 3'T overhang using Taq with only dTTP for a substrate. Again I beleive the protocol is also on OpenWetWare
-perneseblue-
QUOTE (perneseblue @ Nov 10 2006, 05:02 PM)
if memory serves OpenWetWare has a cloning strategy to make a plasmid which contains AhdI sites, that when cleaved produces 3' T overhangs.
another way would be to cut your plasmid with an blunt end enzyme, then add a 3'T overhang using Taq with only dTTP for a substrate. Again I beleive the protocol is also on OpenWetWare
another way would be to cut your plasmid with an blunt end enzyme, then add a 3'T overhang using Taq with only dTTP for a substrate. Again I beleive the protocol is also on OpenWetWare
Ok, I'll look after it, but I still need to attach the Topoisomerase to the 3' ends
-hollegaard-