WinterVomitingLab:Protocols/Partially purified lysate
Return to WinterVomitingLab
Preparation of MNV-1, FCV or HAV partially purified cell culture lysates.
- 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
- 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)
- Pour flask contents into a 50 ml conical centrifuge tube. (Do not fill tubes over 40ml)
- Centrifuge @ 2,000 x g for 15 minutes to pellet cell debris, retaining the supernatant.
- Vacuum filter supernatant using 50 ml conical filter units (membrane pore size = 0.22 um).
- Label tube(s) with contents, the date, and initials, and store at 4°C if you will be using virus right away.
- 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.
Relevant papers and books
- Who has experience with this protocol?
or instead, discuss this protocol.