RT-PCR

RT-PCR for lentivirus tittering
Virus treatment:
1. Add 12 ul virus containing medium to 108 ul lysis buffer (10 mM glycine, 0.5% Triton X-100, pH 2.5);

2. incubate at RT for 10 min;
3. Add 2 ul 1M Tris pH 8.0 to adjust pH to about 7.0;
4. Add 1 ul 10mg/ml DNase;
5. Incubate at RT for 30 min;
6. Incubate at 75 oC for 10min to denature DNase;
7. Use 2 ul to do RT-PCR.

RT-PCR:
2 ul virus sample treated above;
1 ul 10 uM WPRE-F
1 ul 10 uM WPRE-R
12.5 ul 2 X Mastermix
8.5 ul ddH2O
Total 25 ul.

Use ABI7000 to do the RT-PCR.
1. 50°C 2 min;
2. 95°C 10 min;
3. 95°C 15 S;
4. 60°C 1 min;
5. Repeat step 3 and 4 for another 39 cycles.

--Ztao 09:53, 12 April 2011 (EST)