The quick ligation kits use ligase that is 5 u/ul as opposed to the usual 1
u/ul, and the buffer has been optimized for PEG and NaCl concentrations.
There was a paper, I think in NAR where the concentration of these items
was optimized. I have played around with the buffer components and found
that the high Ligase concentration was the most important factor, using 5 u
ligase (1ul) in a 20 ul reaction containing 200 ng total DNA at an
insert:vector ratio between 3:1 and 2:1. 15 minute ligation times worked
fine most of the time.
At 12:30 AM 1/6/00 -0000, David Mckinnon wrote:
>>>We have been using Boehringer Mannheim's rapid ligation kit for some time now
>and find it to work very well. It cuts a day from most cloning procedures and
>gives a very high and reproducible yield of recombinants. Does anyone have a
>home-brew procedure that works as well as the kit? It is unlikely that
>Boehringer has stumbled onto something very novel but our efforts to
>their kit have not worked as well as the proprietary reagents.
>>Thanks for any suggestions