Genomic DNA extraction - Fast extraction method

·         This method is good for organ with highly accumulated phenolic compounds or seedlings.

1.     Materials

·         Homogenization buffer

1 M NaCl

0.2 M sucrose

0.01 M EDTA

0.03 M Tris-HCl, pH 8.0

·         Lysis buffer

0.25 M EDTA

2.5% (w/v) SDS

0.5 M Tris-HCl, pH 9.2

·         Grind buffer (prepare just before use)

4 homogenization buffer + 1 lysis buffer

·         3 M Potassium acetate, pH 4.7

·         70%, 100% EtOH

·         10 mg/mL RNase (Sigma)

·         Pellet pestle (Sigma)

2.     Procedure

a.      Freeze 0.25 g of plant tissue samples in liquid N2, put it to E-tube, and grind with pellet pestle.

b.     Add 400 uL of grind buffer and slightly disturb it at 65ºC (prevent to make any bubbles).

c.      Keep it at 65ºC for 30 min.

d.     Add 13.3 uL of 3 M potassium acetate, pH 4.7

e.      Keep it on ice for 30 min.

f.       Centrifuge it for 10 min at 4ºC, 12,000 rpm and transfer the supernatant to E-tube.

g.     Add 700 uL of 100% EtOH and keep it for 2 min at room temperature (RT). Centrifuge it for 10 min at 4ºC, 12,000 rpm.

h.     Discard supernatant. Wash the precipitant with cold 70% EtOH and centrifuge it for 2 min at 4ºC, 12,000 rpm.

i.       Discard supernatant and dry the precipitant. Dissolve the dried precipitant with 40 uL of ddw and 1uL of DNase-free RNase (10 mg/mL).