I have been trying to ligate my PCR products into the Topo TA vector from invitrogen. I had success the very first time I did the ligation with a fresh PCR product (Not gel purified), but when I repeated the ligation with products extracted from a gel there was no inserts, I tried an A -tailing reaction with no success.
I am getting hundreds of transformants on Lb ampicilin plates but upon analysis with both PCR and restriction digest the plasmids appear to be closing without inserts.. HOW IS THIS POSSIBLE??? It is also reported in this paper http://aem.asm.org/c.../74/5/1649.full that as many as 34% of their transformants were closed plasmids.
So I have two main questions,
1.Does topoisomerase mediated ligation require presence of a buffer like those used in normal PCR to work efficiently?
2.Is there any explanation why my plasmids closing when they have a T over hang on both ends
Also, I am using MyTaq from bioline
Any advice would be greatly appreciated!
Edited by rjoyns, 05 July 2013 - 02:55 AM.