1. Place 510 mg (0.0050.010 g) of finely minced tissue in a 1.5 ml capped tube. Add 300 l xylene and incubate 5 min with constant mixing at room temperature.
2. Centrifuge at 13,00016,000 x g for 13 min to pellet the tissue. Discard the xylene.
3. Repeat steps 1 and 2 twice (for a total of 3 xylene washes).
4. Add 300 l of 100% ethanol to the tube and incubate 5 min with constant mixing at room temperature.
5. Centrifuge at 13,000-16,000 x g for 1-3 min to pellet the tissue. Discard the ethanol.
6. Repeat steps 4 and 5 (for a total of 2 ethanol washes).
1. Add 300 l cell lysis solution, and homogenize using 3050 strokes with a microfuge-tube pestle.
2. Incubate lysate at 65C for 1560 min.
3. If maximum yield is required, 1.5 l proteinase K solution (20 mg/ml) may be added to the lysate. Mix by inverting capped tube 25 times and incubate at 55C until tissue particulates have dissolved (3 hr to overnight). If possible, invert tube periodically during the incubation.
1. Add 1.5 l RNase A solution (4 mg/ml) to the cell lysate.
2. Mix the sample by inverting the capped tube 25 times and incubate at 37C for 1560 min.
1. Cool sample to room temperature.
2. Add 100 l protein precipitation solution to the RNase A-tre a t e d cell lysate.
3. Vortex capped tube vigorously at high speed for 20 sec to mix the
protein precipitation solution uniformly with the cell lysate.