Blunt/Sticky-end ligation

Hiranya S. Roychowdhury hroychow at NMSU.EDU
Tue Sep 7 14:43:52 EST 1999


At 04:36 PM 9/7/99 +0200, Stefan Kappeler wrote:
>I should ligate a 1kb insert into a dephosphorylated 6.5 kb vector. One
>end is sticky (XbaI) the other blunt (PmlI). What are the best reaction
>conditions:
>-temperature, time
>-buffer composition (ATP, PEG,...)
>-molar ratio insert/vector
>
>Many thanks
>Stefan


Simply incubate your ligation rxn at room temp for an hour and transform in
your usual ligation buffer (ie. 1mM ATP, 5% PEG final conc.). 

The molar ratio, IMHO, is unimportant (especially for a single insert). I,
almost routinely, obtain more than 90% recombinants using several fold molar
excess of the insert.


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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