Difference between revisions of "PhiX:Serial dilution"
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==Summary== | ==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 [[PhiX:Plaque assay | double agar plaque assay]] onto which fresh dilutions should be plated. | ||
| − | ''' | + | ==Procedure== |
| − | + | '''Stock Solution'''<br> | |
| − | + | The stock solution has a titer = x PFU/ml (PFU = plaque forming units)<br> | |
| + | Alternatively = y PFU in z ul<br> | ||
| − | ''' | + | '''Serial Dilution Procedure'''<br> |
| − | Add 100ul (10<sup>-1</sup>x) phage to 900ul HFB and mix | + | 1. 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).<br> |
| − | Transfer 100ul (10<sup>-2</sup>x) #1 to 900ul HFB and mix | + | 2. Add 100ul (10<sup>-1</sup>x) stock phage to 900ul HFB and mix -> tube #1<br> |
| − | Transfer 100ul (10<sup>-3</sup>x) #2 to 900ul HFB and mix | + | 3. Transfer 100ul (10<sup>-2</sup>x) #1 to 900ul HFB and mix -> tube #2<br> |
| − | + | 4. Transfer 100ul (10<sup>-3</sup>x) #2 to 900ul HFB and mix -> tube #3<br> | |
| + | And so on... Do not forget to change tips and mix between transfers. <br> | ||
| − | ''' | + | '''Plating'''<br> |
| − | + | Tube #1 -> plate 100ul (1/10) -> 10<sup>-2</sup>x<br> | |
| − | + | Tube #2 -> plate 100ul (1/10) -> 10<sup>-3</sup>x<br> | |
| − | + | Tube #3 -> plate 100ul (1/10) -> 10<sup>-4</sup>x<br> | |
| − | and so on... | + | and so on...<br> |
| + | |||
| + | ==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.<br> | ||
| − | |||
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> | + | x = n / 10<sup>-7</sup><br> |
| + | x = n * 10<sup>+7</sup><br> | ||
OR, if z = 200ul (e.g., mutagenesis)<br> | OR, if z = 200ul (e.g., mutagenesis)<br> | ||
| − | y = (n * 10<sup> | + | y = (n * 10<sup>+7</sup>) * 200/1000<br><br> |
| + | For a diagrammatic version of these calculations see:<br> | ||
| + | [[File:dilution.pdf]] | ||
Latest revision as of 11:06, 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
1. 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).
2. Add 100ul (10-1x) stock phage to 900ul HFB and mix -> tube #1
3. Transfer 100ul (10-2x) #1 to 900ul HFB and mix -> tube #2
4. 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
