genome size and library screening
Mark D. Garfinkel
mg16 at kimbark.uchicago.edu
Fri Sep 15 09:07:50 EST 1995
In article <199509150819.KAA16095 at techunix.technion.ac.il>,
Zeev Lev <zlev at TECHUNIX.TECHNION.AC.IL> wrote:
>>The "ballpark" in our lab for screening genomic libraries of D.
>>melanogaster is to screen 1 x 10^6 plaques (one million) in order to get a
>>fair representative of the genome. What kind of numbers do you employ?
>The genome sizes of D. melanogaster and M. Domestica are 1.65x10^8 and
>8.4x10^8 bp, respectively. To isolate a specific sequence from a library
>with 17 kb inserts (probability of 99%, P = 0.99) you have to screen (only)
>4.5x10^4 and 2.3x10^5 plaques, respectively (1). The appropriate numbers for
>a mammalian library are 3x10^9 bp and 8.1x10^5 plaques (2).
Note that in any library screening there are major assumptions made:
e.g., that average insert size is accurately known, that all the sequence
complexity of the genome was clonable under the circumstances, that all
clones propagate with equal viability (esp. important for library samples
that had been amplified repeatedly, and usually incorrect), that the Poisson
distribution usually employed in the calculation is valid, etc., etc.
While Lev's Drosophila estimate of 4.5e4 clones is approximately the
value I'd calculate, in practice, I just round up to 1e5 lambda clones.
Ten 150-mm plates, each with 1e4 phage. Piece-o-cake, esp. compared to
the Cornell poster's probable ten-fold over-screening.
Mark D. Garfinkel (e-mail: mg16 at midway.uchicago.edu)
(c) 1995; all rights reserved. Permission granted for Usenet quotation
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