Blunt-end cloning

Dickens Lum lumdicks at netvigator.com
Fri Aug 15 10:18:04 EST 1997


Hi Netters,

     I have several questions concerning the blunt-end cloning using
the PCR-Script Blunt-end Cloning Kit from Stratagene.

       I've performed five cloning reactions and the results
are very unsatisfactory . My PCR products (300bp) are generated using
the Pfu polymerase.
It was then electrophoresed and eluted from gel using the Sephaglas
Band-Prep Kit
from Pharmacia. Ligation reaction is then followed exactly as described
in the manual, with an insert:vector = 70:1,  at 27C, using a new batch
of Srf I enzyme just purchased fron Stratagene. Result: 30 ~ 40% of the
colonies are white in color, with about 200 ~ 400 total colonies each
plate. I've picked 30 white colonies (total 150 for five cloning
reactions) for further analysis ( by restriction digestion of the
extracted plasmid) but NONE of them contain the desire insert. ALL of
them are FALSE positive.

        Besides, some of the colonies are lightly blue in centre and
white in peripheral. Are these colonies positive or negative clone..??
In fact, some of the plates have none absolutely white colonies but
these.

        I'm very frustrated with this result and hope you can give me
some suggestions. Thanks in advance...

Regards,
Lum




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