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,
190-193.
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:
7617-7631.


Tim Fitzwater


Principal Research Associate


Gilead Sciences


>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
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?



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