Method A
Method A was the chloroform/acetone method. This
method was performed according to the protocol described
described
by Xie et al. (2007). The powdered sample
was homogenized in 10 mL of extraction buffer [0.1
mol L-1 KCl, 0.5 mol L-1 Tris-base at pH 7.5, 0.05
mol L-1 ethylene diamine tetraacetic acid (EDTA), and
2% β-mercaptoethanol (β-ME)] for 10 min. The mixture
was shaken on ice for 2 h and centrifuged at
12 000 × g for 15 min at 4°C. The supernatant was then
transferred to a new tube and an equal volume of water
saturated chloroform and isoamyl alcohol (24/1, v/v)
was added. The tube was gently shaken on ice for 30
min, then stored on ice for 10 min before centrifugation
at 10 000 × g, for 10 min at 4°C. The upper and
bottom phases were removed. Then 4 mL of water
was added to the interphase together with an equal volume
of water-saturated chloroform and isoamyl alcohol
(24/1, v/v); the mixture was homogenized, incubated
on ice for 10 min, and then centrifuged at 10 000 ×g
for 10 min at 4°C. The treatment was repeated twice.
The interphase was washed with ice-cold acetone 3
times. The final pellets were vacuum-dried and stored
at -80°C.
Method AMethod A was the chloroform/acetone method. Thismethod was performed according to the protocol describeddescribedby Xie et al. (2007). The powdered samplewas homogenized in 10 mL of extraction buffer [0.1mol L-1 KCl, 0.5 mol L-1 Tris-base at pH 7.5, 0.05mol L-1 ethylene diamine tetraacetic acid (EDTA), and2% β-mercaptoethanol (β-ME)] for 10 min. The mixturewas shaken on ice for 2 h and centrifuged at12 000 × g for 15 min at 4°C. The supernatant was thentransferred to a new tube and an equal volume of watersaturated chloroform and isoamyl alcohol (24/1, v/v)was added. The tube was gently shaken on ice for 30min, then stored on ice for 10 min before centrifugationat 10 000 × g, for 10 min at 4°C. The upper andbottom phases were removed. Then 4 mL of waterwas added to the interphase together with an equal volumeof water-saturated chloroform and isoamyl alcohol(24/1, v/v); the mixture was homogenized, incubatedon ice for 10 min, and then centrifuged at 10 000 ×gfor 10 min at 4°C. The treatment was repeated twice.The interphase was washed with ice-cold acetone 3times. The final pellets were vacuum-dried and storedat -80°C.
การแปล กรุณารอสักครู่..
