Sv: blunt-end ligation
tfitzwater at gilead.com
tfitzwater at gilead.com
Mon Nov 6 14:42:59 EST 2000
Historical reasons for performing ligations at 14-16° include:
1) T4 DNA ligase unit definition: 0.015 Weiss unit of T4 DNA ligase will
ligate 50% of the Hind III fragments of bacteriophage lambda (5 µg) in 30
min at 16°C.
2) One of the early references regarding overnight incubations at 16° was
A. Hu BRL Focus 1983 Effect of Temperature on ligation of Hind III-cleaved
DNA 5(3): 13.
3) A subsequent paper (P. W. King and R. W. Blakesley, 1986 Focus 8(1):1-3)
examined the effects of PEG and incubation time and temperature.
4) Also see Bercovich et al. 1992 Effect of DNA concentration on
recombinant plasmid recovery after blunt-end ligation. BioTechn 12:2,
As for the rapid ligation kits that are commercially available, I suspect
that these kits primarily use 5 Weiss Units of T4 DNA ligase per reaction
in a buffer optimized for PEG and NaCl concentrations. Try 5 units of
ligase in a 20 µL reaction using Life Technologies ligation buffer (which
contains PEG), containing 200 ng total DNA for 15 minutes as a substitute.
Possible reference: Hayashi, K. et al., 1986 Nucleic Acids Research 14:
Principal Research Associate
>Actually yes, ligation by thrmocycling works great!
>But for a single fragment ligation 15 min at RT seems to be sufficient. I
>get reasonable result ligating blunts at RT for about an hour.
>At 09:18 PM 10/31/00 +0100, 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
>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
>in virtually all cases.
>Why do the ligation at low temperature, when the ligase has optimum
>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
>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?
>----- Original Message -----
>From: "Dr. Hiranya S. Roychowdhury" <hroychow at nmsu.edu>
>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)
>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.
>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.
>At 10:16 PM 10/26/00 +0100, Rodney Earl Pettway wrote:
>>Anyone have a good blunt-end ligation protocol?
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