Difference between revisions of "GHV-68 Plaque Assay"

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(Procedure)
(Procedure)
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==Procedure==
 
==Procedure==
 
Day 0
 
Day 0
#Trypsinize 3T12's (1 confluent T175 will yield approx. 10-40x10</sup>6
+
#Trypsinize 3T12's (1 confluent T175 will yield approx. 10-40x10<sup>6,\sup> cells) and resuspend at 2.5-2.5x10<sup>6<\sup>/ml in DMEM-10.
#Incubate at 37&deg; C for 20-30 min.
+
#Aliquot 2/ml/well (3-5x10<sup>5</sub> 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.
#Add 0.1 volume resuspended DNase Inactivation Reagent.
+
##Need one 6-well plate per viral sample. (This will let you test 6 logs of dilution)
#Incubate 2 min at RT mixing every 30s.
+
#Prepare[[Methylcellulose]], if necessary.
#Centrifuge at 10,000xg for 1.5 min.
+
#Freeze/thaw/freeze your viral sample, if necessary.
#Transfer solution to a new tube avoiding pelleted Inactivation Reagent.
 
  
 
==Notes==
 
==Notes==

Revision as of 11:36, 30 May 2007

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,\sup> cells) and resuspend at 2.5-2.5x106<\sup>/ml in DMEM-10.
  2. Aliquot 2/ml/well (3-5x105</sub> 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. PrepareMethylcellulose, if necessary.
  4. Freeze/thaw/freeze your viral sample, if necessary.

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. 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

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.