Posted 15 June 2010 - 04:19 PM
-insert digested with EcoRV/BamHI
-vector digested with EcoRV/BamHI and CIP'd
-both gel purified
-ligated with 1:3 vector:insert ratio
-control plate has 0 colonies (vector only)
-ligation plates have 20-30 colonies (vector + insert)
I tested colonies from the ligation plates via test digests and all were negatives. Why then do I get so many colonies with vector + insert as compared to my vector only plates if my colonies all turn out negative (my insert does not drop out when i test digest).
Posted 26 July 2010 - 02:41 PM
if the insert is big and should be able to be seen on a gel, it may sound trivial but the only thing i can think of is to check your selectable marker. it happen to be once that i was using the wrong antibiotic due to some miscommunications about the plasmid and changing the anti-biotic helped.
Posted 28 July 2010 - 07:44 AM