Thank you all for advice!
Because I think the problem is that PCR products with insert cannot be cut well by restriction enzymes, later PCR products with insert was cloned into pGEM-T first, and then white-blue screen, I already got the plasmid with insert (checking by sequencing), afterwards cut the plasmid with NheI and PstI, the new plasmid has one NheI site and two PstI sites, but I think it can not be the problem to get the right insert, I already checked it on the gel.
As far as the digestion of vector was considered, I also check on the gel, it should be no problem.
After that, I do the ligation with different ratio of insert to vector (2:1. 3:1, 9:1), both overnight 4 C and room temper for 2 hours, but I just got one colony on the plate, no colony on control (without insert). When I checked this colony by colony PCR, there is still nothing, all the bands at the bottom of gel! It's so pity!
I already worked on that for 3 months! How come?
Many thanks in advance!
Edited by Biogareth, 09 April 2009 - 05:03 AM.