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Blund End Cloning Fails- Reason ?????

Blund End Cloning Fails- Reason ????? - Protocols and Methods Forum

Blund End Cloning Fails- Reason ????? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 11-08-2005, 11:16 AM
vijaykr
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Default Blund End Cloning Fails- Reason ?????



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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