DNA extraction and electrophoresis

DNA extraction was based on the method of Young and Gallie (2000), which was modified as follows. Thirty axes from seeds subjected to accelerated ageing for different periods of time were homogenized to a fine powder with a mortar and pestle under liquid nitrogen. DNA was subsequently extracted by grinding the powder in 200 |ll of an extraction buffer composed of 10 mM tris- (hydroxymethyl)-aminomethane-hydrochloride (tris-HCl) at pH 8.0, 10 mM NaCl, 10 mM ethylene-diaminetetraacetic acid (EDTA) and 1% (w/v) sodium dodecyl sulphate (SDS). Following this, 50 | l (1.0 mg/ml) protease K was added to the homogenate and incubated at 37°C for 30 min. After incubation, 200 |ll 2% (w/v) cetyltrimethylam-monium bromide (CTAB), 400 |ll chloroform:isopentanol (24:1) and 400 |ll trisphenol were added to the mixture to remove proteins. The mixture was centrifuged at 12,000g for 5 min and then 100 |Ul isopropanol was added to the supernatant. The supernatant was centrifuged at 12,000g for 5 min once again, and the resulting DNA was washed in 70% (v/v) alcohol, air-dried and resolved in 100 |l tris-EDTA buffer. Then 1 |l (1 mg/ml) RNase A was added to the DNA solution to remove RNA.

DNA electrophoresis was carried out according to Young and Gallie (2000). DNA fragments (20 |lg DNA/lane) were separated on a 2% (w/v) agarose gel, followed by visualization by staining with ethidium bromide.

Was this article helpful?

0 0

Post a comment