re ligation trouble

[batc at ac.dal.ca]

In article <1992Sep9.085708.17107 at gserv1.dl.ac.uk>, skspoidn at susssys1.reading.ac.uk writes:
> 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