I try to cloned a 2.8Kb insert, cut AflII-DraIII into a 14kB plasmid, also cut AflII-DraIII for 4 months, without succes.
I have put my ligation mixture on gel, and I can see a shift between my band on my ligation mixture compared to my linearise vector, so I think that my problems don't occured during the ligation step, but after, during the transformation or the selection of the clone on plate.
I have checked the bacteria (SUREcells): they are competents. I did a ligation control with pCR2.1 vector cut with EcoRV and I got about 1000 colonies. I did a ligation with only the vector without insert and I got no colony on plate. I did a transformation with the uncut vector and I got few colonies (about 50) on plate.
So, I'm thinking that the problem is that the vector, because of it 16kb long, is instable in bacteria.
This is the way I proceed to get clone on plate. Transformation of my DNA (10uL) in SUREcells bacteria (100uL), heat sock at 42C 90 sec, add medium without antibotic (1ml) and they grow 90 min at 37C with gently shacking. Then, I spin cells, resuspend in 100 uL of LB medium and plate them on LB-ampi plate. Plates are incubate o/n at 37C. Usually, I get 20 colonies on each plate. Them, I pick up several clone in 3 ml of LB-ampi and they grow at 30C for 16-20 hours without shacking. Following miniprep, all clone have plasmid DNA, but no one have the same uncut patterns, compared to my uncut vector (16,8 kB long).
So, what do I do wrong? Do you have suggestion that can help me? Does it exist an other strategie? I'm at a point that I am ready to try anything, if it could give my good results.
Thanks in advance,
Edited by dan_, 26 May 2010 - 07:29 AM.