Problems with ligation and transformation
Posted 27 May 2011 - 05:29 AM
I have a big problem with ligation and transformation reactions. I have basically 3 types of ligation reactions:
(1) Vector and insert are both cut with EcoRI, vector is CIPed.
(2) Vector and insert are cut with two different enzyme
(3) Vector is cut by one adhesion end and one blunt end, insert was blunted by T4 polymerase and then cut with a second enzyme
However, after ligation and transformation, only the first reaction gave me colonies. But there are equal numbers of colonies in both vector only vs vector plus insert. I miniprep 8 of them, they are all vector self-ligation. I tried 4 degree ligation vs room temp, it's the same result. It seems my ligase is OK, given the fact that vector self-ligation happens, maybe the CIP is not good. But I really have no clue why the other reaction never gave me colonies.
Posted 27 May 2011 - 09:03 PM
Posted 30 May 2011 - 11:29 AM