GHV-68 Plaque Assay

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Plaque assay for gHV-68 using 3T12 fibroblasts. Stolen from Virgin lab protocols.


  • Methylcellulose
  • X μL reagent 2
    • component A (reagent 2 is made up of multiple components)
    • component B
  • equipment 1
  • equipment 2


Day 0

  1. Trypsinize 3T12's (1 confluent T175 will yield approx. 10-40x106, cells) and resuspend at 2.5-2.5x106/ml in DMEM-10.
  2. Aliquot 2/ml/well (3-5x105 cells/well) into 6-well plates. Don't swirl the cells, as you may end up with all your 3T12's in the center of the well.
    1. Need one 6-well plate per viral sample. (This will let you test 6 logs of dilution)
  3. Prepare methylcellulose, if necessary.
  4. Freeze/thaw/freeze your viral sample, if necessary.

Day 1

  1. Thaw your sample that you want to titer and warm methylcellulose to 37° C.
  2. Perform 10-fold serial dilutions in a round-bottom 96-well plate.
    1. Each column represents a different sample of virus to be diluted.
    2. Each row represents a 10-fold dilution of the virus, ie—row A is 100, row B is 10-1, row C is 10-2, etc.
      1. Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.
    3. Aliquot 225 ul/well fresh media in row B-H.
    4. Aliquot 250 ul/well neat virus in row A.
    5. Transfer 25 ul with multichannel pipette from row A to row B.
    6. Pipette up and down 15 times to mix thoroughly the contents of the well.
    7. Change tips.
    8. Repeat for row B to row C, etc.


  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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Relevant papers and books

  1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. PubMed ID:6947258 | HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. PubMed ID:13718526 | HubMed [Jacob-JMB-1961]
  3. ISBN:0879697164 [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed | HubMed


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