bckraev at aeolus.ethz.ch
Wed Dec 14 05:27:11 EST 1994
I have been using GEM-T for nearly two years and have bought probably 5 or 6
kits. I use 3 times more ligase than the manual recommends and usually ligate
for 4-6 hours and then transform XL-1. I do not use X-gal screening, instead
I screen colonies by PCR with two primers which are about 100 bp apart on both
sides of the polylinker. Indeed, I saw some clones, showing deletions, but
not too many. Contrary to company's claims, substantial number of "empty"
colonies contain intact GEM5Zf ( with EcoRV site apparently religated ).
However, I guess that the problem Rae Nishi encounters has something to do with
PCR fragments NOT having correct A-overhangs. If only one end gets ligated,
the linear molecule may get truncated inside the cell and show deletion at
one side of the MCS. Such problems were common about 10 years ago when ligase
preparations were not concentrated enough and blunt end ligations typically
produced clones with deletions at either end of the MCS. Hope this helps.
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