Ligation problem

Mikek kalch at ulam.generes.ca
Sun Jul 23 21:37:47 EST 1995


In article <3uupoh$spv at gate2.internet-eireann.ie>, Robert Henderson
<chisler at internet-eireann.ie> wrote:

> Dear netters,
> 
> I have been having problems ligating fragments excised from agarose 
> gels which have been cleaned using Bio101 Geneclean system.  Four 
> people in our department have encountered the same problem that is 
> no recombinants when we try to ligate (by cohesive ends) into any 
> number of vectors.  This is not a ligase problem since we have even
> bought fresh ligase thinking that this was the problem.  It is 
> apparently a new problem since I have only encountered it now 
> and never remember having a problem with this system in years gone by.
> Anybody got any ideas?
> 
> 
> Cheers

We have had the same problem here Robert.  We tried to get our efficiency
up and we eventually figured out that the problem was two-fold:
1.  All the new-wash must be removed before resuspending the pellet for
elution of the DNA.
2.  We no longer use just TAE gels, apparently the LMP (nuseive) is void
of some inhibitor of ligase, fancy that, uh.?


I bet if you try those two suggestions you will see your efficiency shoot up!!

Let me know how it turns out!

kalch



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