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 replicate
>their kit have not worked as well as the proprietary reagents.
>>Thanks for any suggestions
Indeed, I do all my ligations for 1h at room temp these days. I do not use
any kits for that. I am particular about my vector and insert preps. With
good DNA as the starting material, I perform ligation for 1h (RT) and
transform cells either fresh or frozen comp cells. Colonies appear in about
8-10h (that would be overnight for convenience).
I do not know what the kit contains or what its protocol is, so I cannot
comment on that.
I use the good ol' 5x ligation buffer (Focus, w/ 5% PEG final conc.) or the
5x ligation buffer from BRL (which is the same, I think). The T4 DNA ligase
I prefer is from BRL (1U/uL) and I use 1uL per 10uL rxn. I do the ligation
in 10uL vol in 0.5mL tubes. I have also tried 15min ligation for sticky
ends, and found it to work just fine.
When one needs to ligate a single insert (or double), this quick protocol
should be fine. I would do the ligation for extended time (up to 6h at RT
for blunt and at 15C for sticky) if the objective is to make a library.
Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu