❞ كتاب SV40 Protocols ❝ ⏤ Leda Raptis

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Anatomy
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3.2. SV40 Virus Stock Production
1. Prepare 80% to 100% confluent T75 flasks of BS-C-1 or CV-1 cells (see Note 1).
2. Remove medium from dishes and replace with 2 mL of MEM-2% FBS containing the appropriate dilution of SV40 plaque forming units (PFU’s) to achieve a
multiplicity of infection (MOI) between 0.1 and 0.01 (see Note 2).
3. Allow infection to proceed for 2 h in an incubator while rocking plates at 15-min
intervals to assure complete coverage and even distribution over the monolayer.
4. After this period, MEM-2% FBS should be added to each plate to obtain a final
volume of 10 mL of medium per 10-cm plate.
5. After 4 d of incubation, the medium should be replaced with fresh MEM-2%
FBS. After this point, cells should be checked daily for signs of cytopathic
effects (CPE) by comparing infected monolayers to the uninfected control (see
Note 3 and Fig. 1).
6. When CPE develops to the point of complete destruction of the monolayer with
floating clumps of cells, place flasks at –20°C overnight or until completely frozen and then thaw at room temperature. Repeat for a total of three freeze/thaw
cycles.
7. This is the viral stock. It will contain cellular debris, which can be removed by
centrifugation at 200 RCF for 5 min if desired.
3.3. Titering SV40 by Plaque Assay
1. Prepare serial dilutions of the SV40 virus stock in MEM-2% FBS by putting
20 mL of stock solution into 2 mL of MEM-2 and vortex vigorously (10–2 dilution).
Repeat this procedure using the immediately preceding dilution in place of the
stock to make 10–4 and 10–6 dilutions. 10–7 and 10–8 dilutions should be made by
using 200 mL of the preceding dilutions in 2 mL of MEM-2% FBS (see Note 4).
2. Use 1 mL of each dilution to infect 6-cm dishes of freshly confluent dishes of
BS-C-1 or CV-1 after removing the old medium. Be sure to include a dish with
1 mL of MEM-2% FBS without virus as mock control.

Biology
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Research on biology for the first grade of secondary school
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