Amplifying genomic libraries: How many PFU's per plate?

frist at ccu.umanitoba.ca frist at ccu.umanitoba.ca
Thu Jun 27 17:07:47 EST 1991


I am preparing to screen a pea genomic library in Lambda L47.1. With an 
average insert size of 20kb, approximately 1.2e6 clones are needed to
have a 99% chance of finding a single copy gene.

First, however, I need to amplify the library, since right now I only have
barely enough PFU's to meet the criteria above. The problem is this: when
amplifying, how densely can you plate your phage?

Current Protocols in Molecular Biology (Unit 5.9) recommends plating 2e5
PFU per 150mm petri plate, meaning I could amplify the whole library on six
large plates. Maniatis however, contends that at too high a density, some
cells will be infected with more than one phage, allowing for recombination
between repetitive genomic sequences (the pea genome is almost entirely
repetitive DNA). Maniatis recommends amplifying at a density of 10 -20,000
PFU per 150mm plate. At that density I'd need 60 plates to amplify the
library, and the library would end up in a final volume of 600ml!

I haven't amplified a genomic library before, but I don't recall hearing
about this scale of operation from others who have.

I'd like to hear from those who have screened large eukaryotic libraries.
Is that low plaque density really necessary?

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Brian Fristensky                |  
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frist at ccu.umanitoba.ca          | 
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