Cloning Fails-- Trouble shoot please

ChenHA hzhen at
Tue Nov 8 10:05:50 EST 2005

On 8 Nov 2005 03:21:15 -0800, "vijaykr" <vijaykr at> wrote:

>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

Have you actually done a miniprep, digest and see if it releases a
fragment?  Or sequencing?  It may be that your colony PCR failed for
reasons other than not having an insert.

Did you ligated the EcoRI cut vector (your first control)?  I find it
hard to believe that you only get 3 colonies if you have ligated it. 

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