Cloning Fails-- Trouble shoot please

vijaykr vijaykr at gmail.com
Tue Nov 8 06:21:15 EST 2005


Sorry for the earlier post with such a serious error. I know EcoRI does
not create a blunt end.
This is of coursenot a blunt end cloning. Anyway I am not able to get a
bit success in this.
I have repeated three times this cloning and every time I get numerous
colonies none of which contains any insert. Actually I am trying to
clone a EcoRI cut insert into a vector that is almost 9kb. I check my
clones by colony pcr from the overnight grown single colonies from the
bacterial plates.
This time I set up a control while performing transformation.
I transformed
-vector EcoRI cut and purified, I got three colonies
-vector EcoRI cut, column purified and CIP treated and column purified,

I got 7 colonies
-vector EcoRI cut, column purified and CIP treated and column purified
plus insert EcoRI and purified in the ratio of 1:4 and 1: 6.  I got
more than 100 colonies.


Can anyone explain what went wrong? None of the colonies contained the
insert when I checked them using primers specific for the insert.
However, control pcr worked. Control contained the insert, EcoRI
purified fragement



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