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Ligation problems

Ligation problems - Protocols and Methods Forum

Ligation problems - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-28-2007, 07:55 AM
Virash Gupta
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Default Ligation problems



I would request the answer from molecular biologists working in India.
I am facing serious problems in ligation of sticky ends of DNA
fragmnets in vector with compatible sticky ends. I know the problem is
with Liagse enzyme. I have tried Ligase (5 units/ microlitre) using
1microlitre/ 20 microlitre Rx using different freshly acquired lots
from MBI Fermentas, Bio Lab and also Banagalore genei ( 5 unit/
microlitre preparations). Sometime these worked when used fresh supply
but most of the time failed. Since ligase is thermosensitive is likely
to loose activity, I also tried 400 units/ microlitre preparation from
banagalore Genei- same results. This I am facing for the last three
years. I know, I can handle ligations as during my work at UC, San
Diego, CA USA, while using ligase from Gibco BRL I never faced this
problem. I need to be suggested with a reliable source of ligase for
its consistent performance in India keeping in view that ligase from
other sources might be loosing its activity during transportation.
Dr V K Gupta
[Only registered users see links. ]

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  #2  
Old 07-28-2007, 11:32 PM
Joe Doe
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Default Ligation problems

In article <[Only registered users see links. ].net> ,
"Virash Gupta" <[Only registered users see links. ]> wrote:
I know, I can handle ligations as during my work at UC, San


Since you use the word "might be losing activity" it does not look like
you know that it is loosing activity. Why not monitor a ligation
reaction on a gel? Take something like lambda Hind III and ligate and
run a gel. NEB has a picture of the smear of products you expect in
their catalog. Do this as a standard control for every ligation until
the problem is solved.

Secondly, if you suspect enzyme quality perhaps your restriction enzymes
also leave nibbled ends etc which contribute to your ligation
difficulties.
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