frist@ccu.umanitoba.ca (06/28/91)
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@ccu.umanitoba.ca | Office phone: 204-474-6085 | - Robert A. Heinlein FAX: 204-275-5128 | ===============================================================================