Plaque Assay

Materials
  1. Virus stock
  2. Test cells
  3. LowMeltingPoint agarose
  4. 10% buffered formalin
  5. 0.2% Methylene blue
  6. 6-well plate
  7. Sterile 15 ml tubes
  8. Pipette and tips

Procedure

  1. Plate Test cells on 6-well plate to be 100% confluent at time of infection
  2. Serially dilute virus stock at 10 fold, 6 levels, in 1ml media per dilution.
  3. Remove media from plate and add diluted virus @ 1ml per well.
  4. Allow to attach for 1hr @ 37oC.
  5. Aspirate virus and wash 1X with media.
  6. Overlay with LowMeltingPoint agarose.
  7. To prepare agarose, make a 2.5% stock in H2O. AUTOCLAVE. Microwave to melt prior to use and keep at 45 – 50 ° water bath until use
  8. Make a 1/7 dilution of agarose using media and add 1.5-2 ml per well.
  9. Allow agarose to solidify @ room temperature.
  10. Let plaques form 4-7 days in 37oC incubator.
  11. FIX with 10% buffered formalin in H2O for 20 min.
  12. Stain with 0.2% Methylene blue for 5 min. Wash off with H2O.
  13. Count number of plaques and calculate # of PFU/ml.

 

 

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