Help wanted with blunt ligations (molecular biology)

Aart A. van Apeldoorn A.A.vanApeldoorn at med.ruu.nl
Thu Sep 21 11:38:37 EST 1995


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!

-- 
Aart A. van Apeldoorn
Dept. of Medical Physiology & Sports Medicine
Faculty of Medicine, Utrecht University
MFU-2 "Stratenum"
Universiteitsweg 100
3584 CG Utrecht
The Netherlands
E-mail: A.A.vanApeldoorn at med.ruu.nl



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