1. Rinse cells/constructs with PBS (3x, 5 min ea.)
CONSTRUCTS
2. Transfer constructs to 2 ml eppendorf tube
3. Add 0.5 ml TRI reagent and homogenize
4. Add 0.5 ml TRI reagent to tubes to total 1 ml added
5. Vortex for 15 s. and react 10 min.
CELLS
2. Add 1 ml TRI reagent to cells and react for 5 min
3. Transfer TRI reagent to 2 ml eppendorf tube
4. Vortex for 15 s. and react for 10 min.
ALL
6. Add 0.2 ml chloroform to tubes, vortex 15 s. and react for 10 min
7. Centrifuge at 12000g for 15 min. at 4 ºC
8. Transfer aqueous phase (top) to new eppendorf tube and add 0.5 ml isopropanol
9. Vortex 15 s. and react at RT for 10 min.
10. Centrifuge at 12000g for 10 min. at 4 ºC
11. Remove supernant (carefully) and wash pellet with 1 ml 75% ethanol in 0.1% DEPC water
12. Vortex sample for 15 s. and centrifuge at 7500g for 5 min. at 4 ºC
13. Store samples in 75% ethanol at 4 ºC (up to 1 week)