1.      50-100mg leaf tissue in 1.5 ml eppendorf tube (1 cotyledon for
PCR only)

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
50-100 times)

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

10.  Dry

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)

50ml

100ml

Final concentration

Sorbitol (MW 182.2)

3.19g

6.38g

0.35M

Tris-base (1M)

5ml

10ml

0.1M

EDTA (0.5M)

0.5ml

1ml

5mM

Nuclei lysis buffer:

15ml

30ml

Final concentration

Tris (1M)

10ml

20ml

0.2M

EDTA (0.5M)

5ml

10ml

0.05M

NaCl

5.84g

11.68g

2M

CTAB

1g

2g

2%

Sarkosyl: 5% (w/v)

Microprep buffer:

DNA extraction buffer

25ml

15ml

10ml

Nuclei lysis buffer

25ml

15ml

10ml

5% sarkosyl

10ml

6ml

4ml

Sodium bisulfite

0.2g

0.12g

0.08g