GHV-68 Plaque Assay

Overview

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

Materials

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

Procedure

Day 0

1. Trypsinize 3T12's (1 confluent T175 will yield approx. 10-40x10^6, 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.

• • • Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.

1. 1. Aliquot 225 ul/well fresh media in row B-H.
   2. Aliquot 250 ul/well neat virus in row A.
   3. Transfer 25 ul with multichannel pipette from row A to row B.
   4. Pipette up and down 15 times to mix thoroughly the contents of the well.
   5. Change tips.
   6. Repeat for row B to row C, etc.

Notes

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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References

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. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

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