screening phagemid libraries

Dennis J. Templeton djt2 at po.CWRU.Edu
Fri Sep 11 12:41:34 EST 1992


In a previous article, horton at molbio (Robert Horton) says:

>Greetings Netlanders!
>
>	I have been using replica plating and colony hybridization to 
>screen "micro-libraries" in a plasmid. This seems to be practical for small
>numbers of colonies, but if I decide to try screening over about 1000, I
>want a better way. First I thought I'd make a mini-library in m13, which
>I can screen by plaque lifting. However, this would require that I re-clone
>the insert into my plasmid once I've identified it. Here's my question:
>since my plasmid (pBluescript II) has a single-stranded phage origin (it is
>a "phagemid"), is there some way to screen them as plaques? I figure if the
>helper phage doesn't lyse the bacteria very well all by itself, one might
>be able to get plaques only where the Bluescript clones are...?


We've been using libraries that we shift from lambda form (ZapII) to
phagemid, to ssDNA, and are worried about retaining complexity from one
step to the next.  Frankly, the step I'm most worried about is making the
ssDNA from the phagemid; if some clones grow much faster they will swamp
out the slowgrowers and we will lose complexity.

If we were doing something as simple as colony/plaque lifts, I'd be
satisfied to keep the library in plasmid form, and screen ampR colonies...
that way you are more assured that complexity is retained.

Just one man's opinion...

dennis



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