Sv: blunt-end ligation

Jens Tornoe jens at tornoe.net
Tue Oct 31 15:18:33 EST 2000


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"
>
>--
>ÐÏࡱá
>
>
>Attachment Converted: "c:\eudora\attach\earlpettway.vcf"
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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