Sv: blunt-end ligation

T. Max M. Soegaard tmms at mbio.aau.dk
Mon Nov 6 12:46:00 EST 2000


Yes there is even a publication stating the same:

Increased cloning efficiency by temperature-cycle ligation
Lund,A.H.; Duch,M.; Pedersen,F.S. (1996)
NAR 24 (4),pp. 800-801

-Max

Jens Tornoe wrote:
> 
> In my experience, ligations are subject to a lot of superstition. In my
> current lab, I have tried to convince people that overnight incubation @
> 12-16 C really isn't necessary, but to no avail. Likewise, buying expensive
> Rapid Ligation Kits is shooting way over target IMHO. As long as you don't
> need library efficiency, conventional T4 and RT incubation will do the trick
> in virtually all cases.
> 
> Why do the ligation at low temperature, when the ligase has optimum activity
> at 37 C? I have been told that it is in order to maximize annealing /
> alignment of the fragment ends, but since I have been lucky to obtain
> positives in a double fill-in blunt cloning after 15 min incubation @ 37 C
> with conventional ligase (had to catch the bus), that cannot be the complete
> answer.
> 
> If low temp is good for annealing and high temp is beneficial for the
> ligation, thermocycling could be an idea.
> 
> Any inputs on that matter?
> 
> Jens Tornøe
> NsGene, Denmark
> 
> ----- Original Message -----
> From: "Dr. Hiranya S. Roychowdhury" <hroychow at nmsu.edu>
> Newsgroups: bionet.molbio.methds-reagnts
> Sent: Friday, October 27, 2000 1:03 AM
> Subject: Re: blunt-end ligation
> 
> Assuming you already have good success in sticky-ends, do the ligation at
> RT in a volume of 10uL to 20uL.
> I rutinely do blunt end ligation as follows:
> 
> 5ng vector (dephosphorylated)
> 5ng insert
> 2uL of the 5x ligation buffer (FOCUS; BRL buffer)
> 1 U ligase
> total vol. to 10uL
> 30 min to overnight at RT
> 
> Transform comp. cells (100uL) with 5uL of the rxn.
> Recover at 37 C w/ very gentle shaking for 30-45min.
> Plate.
> 
> If you are not making a library of some sort, this will be just fine.
> 
> I'm in a rush. If you need a more detailed protocol, email me.
> 
> ciao
> 
> Hiranya.
> 
> At 10:16 PM 10/26/00 +0100, Rodney Earl Pettway wrote:
> >Anyone have a good blunt-end ligation protocol?
> >
> >"PEACE"
> >
> >--
> >ÐÏࡱ
-- 
T. Max M. Soegaard

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