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TRANSDUCTION: PHAGE- TITTERING LAB
Transcript of TRANSDUCTION: PHAGE- TITTERING LAB
Viruses that attack bacteria
Two types of life cycle:
The process beginning with
a single phage genome and release
of new phage progeny
2. Lysogenic cycle:
DNA is integrated into the host chromosome
The inserted phage genome or prophage is passively replicated as part of the bacterial chromosome
Grow E. coli to mid-log phase in LB medium containing 5mM Ca2+.
Take phage and serially dilute in the LB + calcium (5mM) from 10-2 to 10-7 .
Take 100 ul of phage from each dilution and mix with 100 ul of E. coli.
Incubate for 20 min at 37OC, but room temp. is OK if necessary..
Rapidly add 2.5 ml of top agar that has been maintained at 47OC and vortex quickly.
PFU/ml=Number or plaques X 10 x dilution factor
=15 X 10 X 106
Pour quickly on an R-plate and swirl the plate while it is on the laboratory bench.
Allow the agar to solidify, and then incubate 16 to 24 hours for plaques to develop.
P1 plaques are small, and can be seen better by holding the plate up to a light.
Count the plaques and calculate a titer. We call this a PFU for plaque forming units.
The plaque assay:
originally a virological assay employed to count and measure the infectivity level of the bacteriophages. But later, it was applied to measure and count the mammalian viruses as well.
Purpose: for the isolation of virus and its purification, and to optimize the viral titers.
Basis: plaque assay is to measure the ability of a single infectious virus to form a “plaque” on a concurrent monolayer culture cells.