library amplification

Dom Spinella dspinella at chugaibio.com
Fri Jul 11 09:48:15 EST 1997


> Hi,
> I need to amplify a cDNA library in Lambda Zap II. The literature that
> came with the library indicates that there were 2 X 10(6) primary
> plaques; I'm assuming that means that I need to amplify 2 million
> phages to ensure complete library amplification. If so, the
> amplification procedure will be quite cumbersome (ie: 40 large plates;
> 8 - 10 ml SM buffer overlays per plate to collect phages = 320 ml phage
> suspension). Is there an easier method? Or have I missed something in
> my interpretation of the amplification procedure? Thanks, Christie

Christie:

Actually, I don't think you need to do plate overlays at all.  You can
amplify your phage in a few liters of liquid broth and it will be far
easier.  Many people believe that plate amplification minimizes bias
resulting from differential growth rates of phage in liquid culture
which theoretically would be minimized on plates (where rate of
infection of new host cells is supposedly limited by rate of diffusion
of phage particles through the agar matrix).  However, my lab has done
experiments which pretty conclusively indicate that there are no real
differences between liquid and plate amplification with respect to
differential growth (albeit for M13 phage, but I would bet for lambda as
well).  Perhaps others in this usegroup would disagree with me, but I
have published data to back up the claim.  See McConnell, Uvegas, and
Spinella, Biotechniques 18: 805-806 (1995) for details. Good luck.  --
D.G. Spinella



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