I digested a plasmid called pUASt(9kb) with NotI(single cut site), dephospharylated it by CIAP(Takara), run it in low-melting agarose and recover it by kit(promega). The insert is 2.5kb which is also digested by NotI. I ligate them using ligase(takara) at 16C, overnight in 25ul. The plasmid is about 20-50ng and insert is about the same weight or more. I did transformation using JM109(takara) whose effeciency is about 10e8.
I always got no colony. Here is the controls:
Plasmid digestion: OK
Other clonings using two different endonucleases:OK
Plasmid self ligation after CIAP:0 colony
Plasmid self ligation without CIAP: around 1000 colonies
Plasmid without CIAP but with insert: arount 100 colonies
Plasmid dephospharylated by 10X diluted CIAP: 0 colony
Can you tell me what further modification should I do, or other controls should I do to find out the problems?
Thank you very much
Edited by mammoth, 22 January 2005 - 10:43 PM.