Plant genomic DNA extraction (large scale) – Using a Proteinase K buffer

·         You’ll get around 50 ug of genomic DNA from 1 g of plant tissue sample.

·         Expensive but effective

·         Quality is enough for a genomic library preparation.

1.     Materials

·         Proteinase K buffer

0.2 M Tris-HCl, pH 8.0

0.1 M EDTA, pH 8.0

1% sarkosyl (Sigma)

100 ug/mL proteinase K (Sigma)

·         70%, 100% EtOH

·         3 M Sodium acetate, pH 4.8

·         CsCl

·         10 mg/mL Ethidium bromide (EtBr)

·         Ddw-saturated 1-butanol

·         TE

·         Dialysis tube

·         Quick-seal tube (Beckman)

2.     Procedure

a.      Add 0.5~1.5 g of plant tissue with 3 mL of proteinase K buffer into a mortar and grind them.

b.     Transfer it to 15 mL conical tube and stay at 45~50ºC for 1 hour

c.      Centrifuge it for 10 min at 3,000 rpm

d.     Transfer the supernatant to a new tube and adjust the volume to 3 mL using a proteinase K buffer

e.      Add 6 mL of EtOH and mix. Add 300 uL of 3 M sodium acetate, pH 4.8 and shake it

f.       Centrifuge it for 15 min at 10,000 rpm (SS-34 rotor) and discard its supernatant. Air drying the precipitant.

g.     Dissolve the dried precipitant in 4 mL of TE. If the precipitant seems to have many contaminants, dissolve the precipitant in 3mL of TE and repeat (e) to (g).

h.     Mix it with 4.5g CsCl and 400 uL of EtBr (10 mg/mL).

i.       Centrifuge it for 16~20 hrs at 53,000 rpm (VTi 65 rotor), 20ºC. If you have VTi 80 rotor, you can centrifuge it for 12~16 hrs at 55,000 rpm or 8 hrs at 73,000 rpm.

j.       Transfer the genomic DNA band to E-tube and add same amount of ddw-saturated 1-butanol. Mix well and keep for 2 min.

k.     Centrifuge it for 3 min at 1,500x g (RT) and transfer the water layer to new E-tube. Repeat for 4~6 times until the pink color from EtBr is disappeared.

l.       Dialyze overnight against 2 liters TE buffer to remove remained CsCl.