Cloning Fails-- Trouble shoot please

Austin P. So (Hae-Jin) nobody at nowhere.com
Tue Nov 8 12:42:42 EST 2005


vijaykr wrote:

> 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

You need to be more specific:
1. size of vector
2. size of insert
3. ligation conditions (%PEG)
4. how many colonies you screened
5. how you quantify vector and insert
6. mass quantity of vector and insert

BTW...CIP is not a magic solution as it tends to chew up overhangs if 
you are not careful...

Cheers

Austin






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