Need experiences for self-ligation - (Mar/19/2006 )

I want to do the self-ligation after enzyme digestion like Mse I. I didn't have experiences on it. Do you have some suggestions for the ligation reaction? Such as DNA concentration, time and temperature. Thanks very much!

liu3zhen

hi
self ligation is done same as ligating with insert.
You digest your plasmid, and add 50ng of it in 10µl reaction volume, with 1µl ligase and 1µl 10X buffer. 1h RT, precipitation, washing, and electroporation/transformation via heat chock... and plating bacteras on appropriate selective medium.

thanks a lot!