Posted 10 March 2005 - 03:18 AM
hi
i was wondring, how did you otained your fragments? Do you mean they are with blunt ends both before the digestion? Is there a possibility that the extremities of these fragments show partial complementarity?
for your ligation reaction, i obtained more efficiency by ligation overnight at 16°. I also have used the quick ligase kit from neb with good results.
For the proportions of your two fragments, i would recommend you to do three assays ; first with an equivalent mass of each fragmentn, and for the two others, consider integrating a fragment in the other. That says for the assay "2" you put 1x mass of 691pb (50ng) and 5x mass 583pb (210ng), and for the assay "3", you put 1x mass of 583pb(50ng) and 5x mass 691pb(296ng).
One of the three should work correctly.
Fred