Blunt-end Ligation

gene cutler genecutl at mendel.berkeley.edu
Mon Sep 18 11:49:12 EST 1995


In article <43jave$8k2 at africa.cis.co.za>, PRETOR at PINELN.AECI.CO.ZA
(PRETORIUS) wrote:

> I am experiencing problems with my blunting reactions - Trying to blunt
> the sticky ends of sites Xba 1 and Nde 1. I tried various kits like the
> Sure-clone blunting kit, Amersham blunting kit and the conventional 
> blunting reactions (Klenow). However, ligations have been performed,
> but did not obtain any transformants.A positive control - Self-ligation 
> was also done in parallel to see if the ligations were working.
> The positive control worked but the ligations of the supposedly
> blunt-ended vector and insert failed.Hence I deduce that the blunting
> reaction did not work in order for a ligation to occur.Any suggestions?

You can always try including some radioactive nucleotides in the fill-in
reaction to see if anything is actually happening.

--gc

...doom is like the handle of a pot,              
it's there, know it, have ice in your            
tea, marry, have children, visit your 
dentist, do not scream at night even    genecutl at mendel.berkeley.edu
if you feel like screaming... -cb     http://sp1.berkeley.edu/gc.html



More information about the Methods mailing list