1. Dissect tissue sample quickly and freeze in liquid nitrogen. Store at -70 to -80C. Fresh tissue may also be used. Work very quickly and keep tissue on ice at all times including when tissue is weighed.
2. Add 510 mg (0.0050.01 g) frozen ground tissue or fresh tissue to a 1.5 ml centrifuge tube containing 300 l cell lysis solution, remove from ice, and homogenize thoroughly using a m i c rofuge tube pestle. Place sample back on ice until next step.
3. Incubate lysate at 65C for 1560 min. Alternatively, 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 for 3 hr to overnight, until tissue particulates have dissolved. 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 Atreated cell lysate.
3. Vo rtex vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate. For samples with high polysaccharide content, incubate on ice for 515 min.
4. Centrifuge at 13,00016,000 x g for 3 min. For samples with high polysaccharide
content, centrifugation at 4C may be required. The precipitated proteins
should form a