help with ligation

john brennand john.brennand at gbapr.zeneca.com
Thu Apr 11 09:58:56 EST 1996


Vic

You can test easily whether the problem is in the ligation by setting up
the following

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 
transformation.

Hope this helps

John

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.





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