WinterVomitingLab:Protocols/Partially purified lysate

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Contents

Overview

Preparation of MNV-1, FCV or HAV partially purified cell culture lysates.

Materials

  • Flask virus-infected cells
    • RAW 264.7 cells for MNV-1
    • CRFK cells for FCV
    • FRhK cells for HAV
  • slow speed centrifuge
  • 0.22 um vacuum filter apparatus

Procedure

  1. Remove frozen flask(s) from freezer and thaw completely. This should be the 3rd of 3 cycles of freeze/thaw after successful virus infection. (see :*Flask infection protocol)
  2. Pour flask contents into a 50 ml conical centrifuge tube. (Do not fill tubes over 40ml)
  3. Centrifuge @ 2,000 x g for 15 minutes to pellet cell debris, retaining the supernatant.
  4. Vacuum filter supernatant using 50 ml conical filter units (membrane pore size = 0.22 um).
  5. Label tube(s) with contents, the date, and initials, and store at 4°C if you will be using virus right away.
  6. For long term storage, aliquot virus stock into 2 ml tubes (labeled with contents, date, and initials) and store at -70°C.
 * MNV-1 and HAV can be stored at 4°C for up to 1-2 months without significant decline in virus titer.
   FCV does not survive as long at 4°C and should therefore be aliquoted and stored at -70°C.

References

Relevant papers and books

  1. []


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