1. I use the qiagen extraction kit for the insert purification
2. The double RE reaction is done with BamHI Buffer as mentioned by NEB in 2 hours with 5units each ( I am also afraid of Star activity )
3. The quick ligation kit NEB, which I incubate at 22.5C in 5 minutes as recommended
4. I also dephosphorylate the vector after RE cutting by CIP.
5. For transformation, I use 100 mcl of DH5a E.coli competent cells
However, so far I coudnot get any colony from the plate !!! I am so upset. I am new in this field, so if you have any idea and experience, please help me
Thanks in advance
Edited by tanaka, 22 August 2004 - 07:04 PM.