Ligation problems

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Sat Jul 28 02:55:55 EST 2007


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
Virashkgupta from gmail.com



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