تحميل كتاب SV40 Protocols PDF - مؤلف أجنبي

تحميل كتاب SV40 Protocols pdf 2001م - 1443هـ نبذه عن الكتاب: 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. .