Failed Ligation - (Jun/15/2010 )

Heres what I have:

-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).

how big is your insert? if it is small like 200bp it may not be visible on a gel in which case you can sequence your plasmid to see if its correct.

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.

This happened to me many times and I never found an explanation for it. Try maximizing your amount of insert. That´s what solved my ligation problems so far. I always use 75 ng of CIP´d vector and fill up the rest with purified insert to a final volume of 15 µl. By the way, is your insert created by PCR? Then cleaning up the PCR product prior to restriction sometimes does the trick.