Help wanted with blunt ligations (molecular biology)

John K. Troyer jtroyer at umabnet.ab.umd.edu
Fri Sep 22 08:02:32 EST 1995


On Thu, 21 Sep 1995, Aart A. van Apeldoorn wrote:

> This is a message for all you molecular biologists over there. I'm having
> trouble with blunt ligations, i'm cloning a 1 Kb fragment into a 8.4 and a
> 4.5 Kb vector. Because of non comparable sticky ends i've blunted these
> ends using klenow DNA polymerase. After blunting i ligate the fragment and
> vector using T4-ligase in a volume of 10ul using 1 unit enzyme. After
> transformation there are just a few or no colonies growing, my efficiency
> is ok and it appears that there is no selfligation (after testing). Can
> onyone give me some suggestions what is wrong here.    Thanks!


I have had good success (well, I guess good is not the correct word for 
blunt end ligations), let me say, better success with my own blunt end 
buffer formulation as compared to the ligase buffers which come from the 
supplier.  I have found that the addition of both BSA and spermadine help 
to stabilize the DNA and this buffer has always given me more consistent 
ligations.  I usually do my blunt end ligations at 16 C. overnight or for 
24 hrs. 

10 X Blunt End Buffer
+++++++++++++++++++++
250 mM Tris, pH 7.4
50 mM MgCl2
50 mM DTT
2.5 mM Spermadine
10 mM ATP
100 ug/ml BSA
++++++++++++++++++++++

Always store the buffer in small aliquots so you don't freeze thaw it too 
many times.  Also, the buffer should smell strongly of DTT.  If it does 
not, throw it away and get a fresh aliquot.


Hope this helps.

John



******************************************
John K. Troyer, PhD
University of Maryland School of Medicine
Department of Biological Chemistry
(410) 706-7518
jtroyer at umabnet.ab.umd.edu
*******************************************



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