Ligation

[ChenHA]

DK wrote:
> In article <1183165644.85275.0 from iris.uk.clara.net>, ChenHA <hzhen from freeuk.com> wrote:
> 
>> in 
>> fact, too high a concentration of DNA is bad for ligation - they tend to 
>> form concatemers rather than circularised plasmids.
> 
> Depends. I recently had to ligate 11 kbp vector (directional 
> cloning but one of the sites was 1-base proteruding 3') with 
> 6.5 kpb insert. The only way I could get it to work was to 
> increase DNA concentration to the ridiculous-sounding 
> 20 ng/ul vector and 180 ng/ul insert.  (In case you wonder, 
> various attempts to use PEG at "normal" DNA concentrations
> did not result in successful ligation). 

It doesn't sound too bad.  When you consider molar concentration, the 
vector concentration is probably well within the range recommended.  Not 
sure why you need to use so much insert though.  I remember there was a 
paper for calculating the theoretical optimal concentration for 
ligation, does  anyone has that reference?

The main problem with large vector is that the transformation efficiency 
falls with increasing size.  True for chemical transformation - I think 
it falls quite steeply for those bigger than 8-9 kbp (if I remember the 
graph right that is, can't swear to that), is it also true to 
electroporation?



> 
> DK