1. Add 300-450 mg dried tissue (finely ground) or 6001,200 mg frozen tissue (which may be finely ground with a mortar and pestle in liquid nitrogen), or 6001,200 mg fresh tissue to a 50 ml tube. Work quickly and keep tissue cold to minimize DNase activity. Note: it may be necessary to vary the amount of starting material depending upon the species, age, tissue preparation, and genome size.
2. Add 18 ml cell lysis solution to the leaf tissue. For dried tissue, cap the tubes and vortex 13 sec to wet the tissue. For unground tissue, homogenize using 3050 strokes with a tube pestle.
3. Incubate cell lysate at 65C for 60 min. After 30 and 60 min, invert capped tube 10 times.
RNase Treatment (Optional)
1. Add 90 l RNase A solution to the cell lysate.
2. Mix the sample by inverting the capped tube 25 times and incubate at 37C for 15 min.
1. Cool sample to room temperature.
2. Add 6 ml protein precipitation solution to the lysate.
3. Cap the samples and vortex vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate. Alternatively, invert a rack containing the samples 150 times (approximately 2 min) to mix the protein p recipitation solution uniformly with the cell lysate. For species with high polysaccharide content, it may be necessary to incubate sample on ice for 515 min.
4. Centrifuge at 3,000 x g for 10 min. The precipitated proteins should
form a tight, green pe