Difference between revisions of "PhiX:Serial dilution"
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| − | 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 | + | 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 [[PhiX:Plaque assay | double agar plaque assay]] onto which fresh dilutions should be plated. |
'''Stock Solution'''<br> | '''Stock Solution'''<br> | ||
Revision as of 06:08, 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.
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...
Maths
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
