re ligation trouble

skspoidn at skspoidn at
Wed Sep 9 04:01:35 EST 1992

re SphI ligations:

have you tried sacrificing a chicken under a full moon? :)

Seriously, 3 small suggestions, because 
I have difficulty with 3' overhangers as well

1) You don't mention if the alk phos treatment worked. I find that 3'
 overhangs are much harder to dephos than 5' or even blunt.

2) What is the composition of your ligase buffer? We recently changed 
to Promega and were horrified to discover that the buffer as supplied
 contains no condensation agents (usu glycerol or PEG), and that these
 agents should be added by the user if neccessary. You could try upping 
these components to max levels and see if that helps.

3) Without wishing to appear arrogant, condescending or patronising,
 have you ensured that your ratio of insert to vector is appropriate?

Hope this helps,

Mike Poidinger
Dept of Microbiology
University of Reading

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