long insert DNA TA clone problem,help me ! urgent! - (Mar/14/2006 )

Hi ladies and gentlemen:

Recently, I ligate a long fragments(4.3kb) to the pGM-T vector(T-Easy Clone vector, there is a T-overhang on each end). I encountered a problem that I nearly cannot get the positive clone after the Blue/white clone screening. All the white clone I picked on the plate is false of fake positive clone. the ligation system: 50ng Vector, 100ng Insert, ligase (from NEB),1ul, and 10X buffer 2ul, add dd-water to 20ul. the place the tube at 4C for more than 36hr. By the way,I purify my DNA from the gel with GeneClean Spin Column Kit, then stored the purifed DNA which is disolved in ddH2O in -20C for about 20 Days without repeated thawing and freezing. How can I succeed ligate the insert to the vector?

Many thanks in advance!

I can only give you tips, I don't have the solution. Keep in mind that your insert is big for a 3 kb vector, it is not impossible, but not very efficient.
I don't know what kind of fragment it is (restriction, PCR), but you could try to do an A-tailing on your fragment.
I always stick to the rule ((ng vector x kb insert)/ kb vector ) x 3 = ng insert and than you would have to add more DNA.
Try to use new pGEM-T. The fabricant denies it, but there are some stories that the T's fall off when the vector is older. And also thawing and freezing of the vector is not good for the T-overhang. Try to use the ligase and ligase buffer in the same kit as the pGEM-T. With a larger fragment you could ligate o/n at 4C.
Good luck!