Sv: blunt-end ligation

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Fri Nov 3 22:32:47 EST 2000


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?
>>
>>"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
>
>
>---
>
>
>
>
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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