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

frist at frist at
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?

Brian Fristensky                |  
Department of Plant Science     | Freedom begins when you tell Mrs. Grundy
University of Manitoba          | to go fly a kite.
Winnipeg, MB R3T 2N2  CANADA    |  
frist at          | 
Office phone:   204-474-6085    |   - Robert A. Heinlein
FAX:            204-275-5128    |

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