white colonies have no inserts, why?

beisz at SUN5.IBP.AC.CN beisz at SUN5.IBP.AC.CN
Thu Jul 16 06:08:06 EST 1998


I made a library of an unidentified DNA, using pGEM-3Zf(+) as vector, EcoRI
and PstI as
cloning site, and E. coli JM109 as host. Two thirds of the transformants are
white, but only
5% of these white colonies contain inserts. Who can tell me why?

You may post here, or mail to beisz at ibp.ac.cn.

The detailed procedure:

1) cut the vector with EcoRI and PstI, recover the large fragments fron gel.
2) cut the exogenous DNA with EcoRI and PstI, phenol-chloroform extraction, EtOH
precipatation.
3) sticky-end ligation, 15-25C, overnight.
4) transformation.
5) isolation plasmids with alkaline method, enzyme cut, electrophoresis.
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