Genomic DNA extraction – using a CTAB buffer

·         You’ll get a relatively clean genomic DNA from a sample with phenolic compounds or polysaccharides using this method.

1.     Materials

·         2x CTAB buffer

2% (w/v) CTAB (cetyltrimethylammonium bromide) (Spectrum Chemical Mfg. Corp., Sigma)

100 mM Tris-HCl, pH 8.0

20 mM EDTA, pH 8.0

1.4 M NaCl

1% polyvinylpyrrolidine (mw 40,000) (Sigma)

·         CTAB precipitation buffer

1% (w/v) CTAB

50 mM Tris-HCl, pH 8.0

10 mM EDTA, pH 8.0

·         1x CTAB

Dilute 2x CTAB

·         TE

·         CsCl solution

10 g CsCl/10 mL TE

·         10 mg/mL EtBr solution

·         20x SSC saturated 2-propanol

Using a upper part when you mix 100 mL of 2-propanol and 50 mL of 20x SSC

·         Chloroform

·         70%, 100% EtOH

·         20x SSC

2.     Procedure

a.      Preheat 2x CTAB and 1x CTAB in 65ºC water bath. Wash a plant sample with ddw and remove an excessive water using a paper towl.

b.     Freeze 10 g of fresh tissue with liquid N2 and grind with pestle and mortar.

c.      Transfer the powder into centrifuge tube.

d.     Add 1 mL of preheated 2x CTAB buffer per gram of powder and mix them.

e.      Add 4 mL of preheated 1x CTAB buffer per gram of powder and mix them. And keep it in 65ºC water bath for 5 min.

f.       Add 10 mL of chloroform and mix them well.

g.     Centrifuge it for 5 min at 12,000 rpm, 4ºC.

h.     Transfer the supernatant to new tube.

i.       Add 2 volume of CTAB precipitation buffer and mix them briefly.

j.       Keep it for 30 min at RT.

k.     Centrifuge it for 5 min at 12,000 rpm, RT

l.       Discard the supernatant and dry the precipitant briefly.

m.   Add 4 mL of CsCl solution per 5 g of sample and dissolve it (it may take more than 1hr).

n.     Add 0.1 mL of EtBr and mix them briefly.

o.     Transfer the solution into ultracentrifuge tube and seal it.

p.     Ultracentrifuge it for 6hrs at 65,000 rpm or overnight at 55,000 rpm at 15ºC.

q.     Pick up the DNA band using a syringe and transfer it to E-tube.

r.       Add 0.5 mL of 20x SSC saturated 2-propanol and mix them. Transfer the clear bottom part.

s.      If the DNA band is strong, add 2x volume of ddw and 8x volume of cold 95% EtOH into (p) and mix them.

t.       After the ultracentrifuge, wash the precipitant with cold 70% EtOH.

u.     Discard supernatant and dry the precipitant (DNA).

v.     Add 0.5 mL of TE buffer and dissolve it.

w.   You can dialyze twice times against 1 liters TE buffer per hour to remove remained CsCl instead of EtOH precipitation.

x.     Collect DNA into E-tube using a membrane bag.