white colonies have no inserts, why?

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Thu Jul 16 09:30:10 EST 1998


In article <9807161054.AA07211 at sun5>,
  beisz at SUN5.IBP.AC.CN wrote:
> 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.
> _____________________________________________________________
>
>

I suspect what is happening is that you are getting a self-ligation of the
vector.  PstI has a 3' overhang and EcoRI has a 5' overhang; it could be that
ligase is treating this as a "blunt-type" self-ligation, and then the bugs
fill in the gap after transformation.  This ligation will not maintain the
Lacz-alpha coding frame, so the colonies will be white. Anyway, you got your
insert, so what is the problem?

Nick Theodorakis
Georgetown Univ. Medical Center

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