Difference between revisions of "PhiX:Serial dilution"
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and so on...<br> | and so on...<br> | ||
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| + | 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.<br> | ||
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E.g. count n plaques on plate from 10<sup>-7</sup>x plate:<br> | E.g. count n plaques on plate from 10<sup>-7</sup>x plate:<br> | ||
x = n * 10<sup>-7</sup><br> | x = n * 10<sup>-7</sup><br> | ||
Revision as of 12:03, 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).
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
OR, if z = 200ul (e.g., mutagenesis)
y = (n * 10-7) * 200/1000
For a diagrammatic version of these calculations see:
File:Dilution.pdf
