Ligation

Deitiker, Philip R via methods%40net.bio.net (by pdeitik from bcm.tmc.edu)
Wed Jun 20 18:21:46 EST 2007


I caught this quote in a message but I don't
have the author. 
 
"
Almost all ligation failures are really failures in preparation of the
DNA, not in ligation, and not in calculating or varying vector:insert
ratios.  The ligation reaction is remarkably forgiving, especially for
cohesive ends.  If you are having trouble, look at the restriction
digests, the treatment of the DNA following restriction, the
purification of DNA following PCR, the overhangs of the cut site on
PCR primer addtion of cut sites, UV damage to DNA during gel
excision, and only rarely at the ligation itself.
"
 
I am trying to clone several fragments into a derivative of E4276
a derivative that has a BamH1 - - - - EcoR1 cloning site. 
 
I have had successful ligations in the past, I managed to make
several clones of the nascent vector and they cut true to the 
restriction sites. 
 
But I have had no success cloning 6 Bgl-II -Insert -- EcoR1 constructs
into this vector. Even following protocols suggesting 10 fm of vector
and 30 fm of Insert, all I get back are reclosed vectors. 
*Bgl-II has compatible sticky ends. 
 
Originally the situation was very bad, many vector colonies, but
I worked on the digestion to reduce the number of colonies to
about 1/10th, approximately 10 vector closures per 10fm of DNA. 
 
The ends of the Insert contain 
C-A-C-G-G-A-G-A-T-C-T-T-G-G - - - - - 
 
According to NEB this should be enough overhang. 
as this
. . .  T-C-C-A G-A-T-C-T-T-C-C - - - - -  cut to 100% 
 
The EcoR1 site is the same thing 
   C-G-G-G-A-A-T-T-C-A - - - - - - - - - - (My insert)
 
t-c-g-a-G-G-A-A-T-T-C-C - - - - - - - - (NEB, lower case = single stranded overhang.) 
 
 
The other issue is transformation. Many here say use 1 ul of DNA for transformation. 
 
I don't know if this works or not, but I have linear increase in clones
from 1,2,4,8 microliters. More than 8 uls of ligation mixture did not result
in more clones and eventually clones declined. 
 
So my thinking here is that if 1 ul is adequate my ligation is really tanking. 
The only transformations are the vector, somehow unclosed after hours
of digestion, near complete digestion in 1 hour with either enzyme (when tested
independently and CIP for several hours. 
 
The vector is grown, Minipreped, restriction digested (monitoring timepoints
by gel) CIP, promega SV PCR cleanuped, prior to reaction. 
 
As for the Insert. 
After restriction digest I clean the DNA up with gene clean turbo, more recently
because the mid range ligation is 30 fm I did a EtOH precipitation completely
dried the DNA and resuspended with 1/10X warm ligation buffer. So there should
this is now three purifications removed from the last Gel. Still no success. 
 
Is it worth making new primers and starting from scratch? is it a common primer
that a certain sequence of overhand works and a very similar overhand sequence does not?
 
Thanks in advance. 
 
 
 
 


More information about the Methods mailing list