Difference between revisions of "PhiX:Plaque assay"
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'''Media Preparation'''<br> | '''Media Preparation'''<br> | ||
| − | The usual media for plating is lysogeny broth (LB). Remember that this is not a defined media (so you should attempt to minimise batch effects for reproducibility) and it comes in Lennox and Miller varieties. The appropriate type is Miller which contains 10g/L of sodium chloride (versus 5g/L in Lennox). Both plate (base layer) and top agars should be prepared. For one litre: | + | The usual media for plating is lysogeny broth (LB). Remember that this is not a defined media (so you should attempt to minimise batch effects for reproducibility) and it comes in Lennox and Miller varieties. The appropriate type is Miller which contains 10g/L of sodium chloride (versus 5g/L in Lennox). Both plate (base layer) and top agars should be prepared. For one litre:<br> |
| − | 10 g Tryptone | + | 10 g Tryptone<br> |
| − | 5 g yeast extract | + | 5 g yeast extract<br> |
| − | 10 g NaCl | + | 10 g NaCl<br> |
| − | 15g agar (if plate agar) | 7g (if top agar) | + | 15g agar (if plate agar) | 7g (if top agar)<br><br> |
Make up to 1 litre in distilled water and autoclave. After autoclaving:<br> | Make up to 1 litre in distilled water and autoclave. After autoclaving:<br> | ||
| − | CaCl<sub>2</sub> to a final concentration of 2 mM, | + | CaCl<sub>2</sub> to a final concentration of 2 mM,<br> |
| − | MgCl<sub>2</sub> to a final concentration of 10 mM. | + | MgCl<sub>2</sub> to a final concentration of 10 mM.<br> |
| − | (for these use hydrated crystals as anhydrous powders do not dissolve effectively). | + | (for these use hydrated crystals as anhydrous powders do not dissolve effectively).<br> |
| − | |||
'''Plating Procedure'''<br> | '''Plating Procedure'''<br> | ||
Revision as of 07:34, 20 February 2017
Summary
This protocol is usually used in conjunction with serial dilution of a stock solution of phage for enumeration of phage titer. But it can also be used with any phage-containing input. With thanks to the Wichman lab for setting me up with a good protocol for this early on.
Media Preparation
The usual media for plating is lysogeny broth (LB). Remember that this is not a defined media (so you should attempt to minimise batch effects for reproducibility) and it comes in Lennox and Miller varieties. The appropriate type is Miller which contains 10g/L of sodium chloride (versus 5g/L in Lennox). Both plate (base layer) and top agars should be prepared. For one litre:
10 g Tryptone
5 g yeast extract
10 g NaCl
15g agar (if plate agar) | 7g (if top agar)
Make up to 1 litre in distilled water and autoclave. After autoclaving:
CaCl2 to a final concentration of 2 mM,
MgCl2 to a final concentration of 10 mM.
(for these use hydrated crystals as anhydrous powders do not dissolve effectively).
Plating Procedure
Add 100ul
Recommended Reading
I recommend checking:
Kropinski, A.M., Mazzocco, A., Waddell, T.E., Lingohr, E. and Johnson, R.P., 2009. Enumeration of bacteriophages by double agar overlay plaque assay. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, pp.69-76.
for more details of the plaque assay and variations thereon.
