Difference between revisions of "PhiX:Serial dilution"

Revision as of 11:05, 20 February 2017

Summary

This protocol describes how to prepare serial dilutions in order to enumerate phage from a stock solution. Before you begin you should also prepare for the double agar plaque assay onto which fresh dilutions should be plated.

Procedure

Stock Solution
The stock solution has a titer = x PFU/ml (PFU = plaque forming units)
Alternatively = y PFU in z ul

Serial Dilution Procedure
Prepare a set of labelled tubes from #1 to #8. Fill each tube with 900ul of HFB (or PBS will suffice and we sometimes add DMSO as a cryoprotectant for immediate freezing).
Add 100ul (10^-1x) stock phage to 900ul HFB and mix -> tube #1
Transfer 100ul (10^-2x) #1 to 900ul HFB and mix -> tube #2
Transfer 100ul (10^-3x) #2 to 900ul HFB and mix -> tube #3
and so on...Do not forget to change tips and mix between transfers.

Plating
Tube #1 -> plate 100ul (1/10) -> 10^-2x
Tube #2 -> plate 100ul (1/10) -> 10^-3x
Tube #3 -> plate 100ul (1/10) -> 10^-4x
and so on...

Arithmetic

The number of plaques on any given plate can be used to back-calculate the titer of the original stock. You should choose plates with plaque counts between 30 and 300.

E.g. count n plaques on plate from 10^-7x plate:
x = n / 10^-7
x = n * 10⁺⁷
OR, if z = 200ul (e.g., mutagenesis)
y = (n * 10⁺⁷) * 200/1000

For a diagrammatic version of these calculations see:
File:Dilution.pdf