pGM-T vector problem - (Mar/16/2007 )
Recently, I ligate a fragments(400bp) 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 blue/white clone I picked on the plate is false of fake positive clone.I purify my DNA from the gel with GeneClean Spin Column Kit.
How can I succeed ligate the insert to the vector?
Didn’t forget to A-tail insert, or fill in the overhangs if you used enzyme/s to get 400bp insert?
Try EtOH precipitation after ligation to get rid of all rubbish before transformation. That will leave only ligated vector.
Check antibiotic. Grow some untransformed cells.
how old is the vector? If it's old or badly handled/stored the overhangs will degrade and you will start getting heaps of re-ligated vector...
Don't simply believe the blue/white selection. Sometimes it will give you false results. Try to identify as many as you can.
Set up a negative control with only pGEM-T vector alone for your transformation, and see what happens. If you got same number of colonies as your other plates, you would presume something wrong like re-ligation of your vector.
Generally pGEM-T vector works greatly, i never got any problem with it, as long as it is fresh.