RFD: Plasmid re-ligation without ligase?

Jennifer Lyon jennyann at ix.netcom.com
Fri Apr 25 09:32:45 EST 1997

In <5jipvu$jhd$2 at sparcserver.lrz-muenchen.de>
salger at wap1.zi.biologie.uni-muenchen.de (Klaus Salger) writes: 

>as far as I remember I read about a system called "Ligation
>Independent Cloning System". I don't remember the details but I think
>it relies on in vivo ligation (and recombination?).

In Ligation Independent Cloning, you use a much longer overhang than
traditional restriction site cloning. Novagen's LIC vectors have 12-14
bp overhangs. Special primers (which include the 'LIC site') are used
to PCR-amplify inserts, which are then treated with T4 DNA polymerase
in the presence of only one dNTP so that the polymerase chews back
until it hits a specific site and then pops in the one available
nucleotide and stops digesting. This leaves you with a 12-14bp overhang
complimentary to the one in the prepared vector. Insert and vector are
then annealed to each other, resulting in a complex that is stable
enough to be transformed effectively into e. coli. The breaks are far
enough apart that they are treated as single-strand nicks and are
'healed' (re-ligated) within the e. coli cells. So in effect, this does
involve in vivo ligation, and it works quite well.

Novagen Technical Services
JennyAnn at ix.netcom.com                  Jenni10647 at AOL.com
"I'm in charge of finding myself and I make sure it never happens."
Methos  in  HL - "Methos"     PWFC:"This is very, very weird."

More information about the Methods mailing list