GHV-68 Plaque Assay

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(Procedure)
(Procedure)
Line 25: Line 25:
##Each column represents a different sample of virus to be diluted.
##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.
##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.
+
*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 225 ul/well fresh media in row B-H.
##Aliquot 250 ul/well neat virus in row A.
##Aliquot 250 ul/well neat virus in row A.

Revision as of 14:44, 30 May 2007

Contents

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. 10-40x106, 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.
  • Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.
    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.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Jacob-JMB-1961]
  3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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