T4 ligation problems with adapters - (Aug/24/2007 )
I am trying to ligate together two small pieces (20bp+ 40bp) of synthetic DNA with T4 ligase from NEB. The 40bp strand results from a digestion which looks complete on a gel. The 20 bp strand (adapter) has the complementary sticky end, and the appropriate synthetic oligo is phosphorated. I have tried the following ratios of 5:1, 3:1, 2:1, and 1:1 of adapter to 40bp strand with no ligation (16C 1 - 2 hours) with where there are 20 pmoles of 40mer in 100 ul and enzyme concentrations of 5U up to 1200U. The problem appears to be that two 20mers are ligated together instead of the 20+40mer construct. Does anyone have any ideas? After a week of troubleshooting, I would appreciate any help/advice. Thanks!
I think we have similar problems. I'm trying to ligate two different double-stranded adapters to unknown sequences in DNA with matching sticky ends. As the adapter mediated PCR is not working, I wonder whether the ligation has been successful! Did you already find a solution... Please let me know!