Sep 22, 2019

Public workspacePlaque assay

DOI
https://dx.doi.org/10.17504/protocols.io.7i8hkhw
Plaque assay
  • Marijn Ceelen1
  • 1iGEM Wageningen
  • iGEM Wageningen 2019
Marijn Ceelen
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DOI: https://dx.doi.org/10.17504/protocols.io.7i8hkhw
Protocol Citation: Marijn Ceelen 2019. Plaque assay. protocols.io https://dx.doi.org/10.17504/protocols.io.7i8hkhw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 22, 2019
Last Modified: September 22, 2019
Protocol Integer ID: 27968
Keywords: Plaque Assay, phage, lambda, T7, plaque assay, phage lamba, plaque, counting plaque, assay
Abstract
This is a protocol for the quantification of phage lamba and T7 titers by counting plaques.
Materials
MATERIALS
ReagentLiquid LB medium
ReagentLB agar
Troubleshooting
Streak LB plate with E. coli (strain should susceptible to phage lambda. Strains that include a prophage lambda are likely to be resistant. Strains LE392 and DH10B have been used with this protocol) and incubate overnight at Temperature37 °C

Pick a colony from this plate and use it to inoculate Amount10 mL of LB. Incubate this culture at Temperature37 °C until OD reaches 2-3. Overnight culture is recommended.

Make a range of dilutions . Mix Amount100 µL of phage dilution with Amount200 µL of E. coli culture. Then incubate for 10 minutes at room temperature. After Duration00:10:00 add Amount3 mL of liquid soft LB agar (LB agar with 0.7% agar) (Temperature50 °C ).



Pour the resulting mixture on LB plates (preheat plates at Temperature37 °C ). Spread the mixture on the plate by moving it. Make sure to work quickly to avoid clumps of solidified agar.

Incubate for Duration00:15:00 at room temperature. Afterwards turn the plates over and incubate overnight at Temperature37 °C .

Use plates with 30-300 plaques to determine phage concentration. Calculate the Plaque Forming Units (PFU)/mL by the following formula: PFU/mL = 𝑁 × 1/𝐷𝐹 × 1/V.
N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the volume of phage dilution poured on the plate.