1. 50-100mg leaf tissue in 1.5 ml eppendorf tube (1 cotyledon for
2. Prepare fresh microprep buffer, RT
3. Add 200ul buffer, grind tissue (rinse pestle with water between
samples). Add another 55ul buffer, shake entire rack by hand.
4. 65C, 30-120min
5. Fill the tube with chloroform, mix well (shaking up and down
6. 10000 rpm, 5min
7. Pipet off aqueous phase (~0.5ml), add 1X volume of cold
isopropanol, invert tube repeatedly until DNA precipitates.
8. Immediately spin at 10000rpm for 5min (No more)
9. Wash pellet with 70% ethanol
11. Resuspend in 50ul TE at 65C for 15min
12. Spin 10min at 10000rpm, store at -20C.
13. 1ul for PCR, 15-25ul for southern blot (5-10ug DNA, 15-20 units
enzyme) (If 1 cotyledon was used, 5ul for PCR)
DNA extraction buffer(pH 7.5)
Sorbitol (MW 182.2)
Nuclei lysis buffer:
Sarkosyl: 5% (w/v)
DNA extraction buffer
Nuclei lysis buffer