help with ligation
john.brennand at gbapr.zeneca.com
Thu Apr 11 09:58:56 EST 1996
You can test easily whether the problem is in the ligation by setting up
1) vector alone
2) insert alone
3) vector + insert (1:3 & 1:10)
All + T4 ligase & also duplicate set without ligase.
( with both +/- phosphatased vector ?)
Then, run a few microlitres of each reaction on a minigel and look for
the ligated DNA to move to higher molecular weight compared to the
reactions without ligase.
If you see the expected pattern in the V+I ligation - go to the
Hope this helps
Oh afterthought, we always use Boehringer T4 and I notice that often the
buffer has "bits" in it that have dropped out of solution - it then
takes some warming before going back in.
More information about the Methods