GHV-68 Plaque Assay
Plaque assay for gHV-68 using 3T12 fibroblasts. Stolen from Virgin lab protocols.
- X μL reagent 2
- component A (reagent 2 is made up of multiple components)
- component B
- equipment 1
- equipment 2
- Trypsinize 3T12's (1 confluent T175 will yield approx. 10-40x106, cells) and resuspend at 2.5-2.5x106/ml in DMEM-10.
- 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.
- Need one 6-well plate per viral sample. (This will let you test 6 logs of dilution)
- Prepare methylcellulose, if necessary.
- Freeze/thaw/freeze your viral sample, if necessary.
- Thaw your sample that you want to titer and warm methylcellulose to 37° C.
- Perform 10-fold serial dilutions in a round-bottom 96-well plate.
- Each column represents a different sample of virus to be diluted.
- 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.
- Aliquot 225 ul/well fresh media in row B-H.
- Aliquot 250 ul/well neat virus in row A.
- Transfer 25 ul with multichannel pipette from row A to row B.
- Pipette up and down 15 times to mix thoroughly the contents of the well.
- Change tips.
- Repeat for row B to row C, etc.
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
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Relevant papers and books
- 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.
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56.
- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. ISBN:0879697164
- Who has experience with this protocol?
or instead, discuss this protocol.