Genomic DNA Extraction
Small-scale extraction of genomic DNA from Arabidopsis leaves was achieved by using a variation of the Dellaporta protocol (Dellaporta et al., 1983). Frozen leaf tissue is first ground to a fine powder in an Eppendorf. To this is added 750ml of extraction buffer (100mM Tris pH 8.0, 50mM EDTA pH8.0, 500mM NaCl, 10mM b -mercaptoethanol); and 50 m l of 10% SDS (to dissolve the cell membranes).
After incubation at 65°C, 250 m l of 5M potassium acetate is added. This is incubated on ice for 20 min; the solution is then separated by centrifugation (14000 rpm for 10 min) (Eppendorf microcentrifuge). 500 ml of isopropanol is added to the supernatant, which is incubated for 20 min at –20 °C. After further centrifugation (14000 rpm for 10 min) (Eppendorf microcentrifuge), the supernatant is discarded and the genomic DNA pellet dried in a drying centrifuge for 5 seconds. The pellet is re-dissolved in 100 m l of 10mM TrisHCL or DH 2O and stored at -20 oC until required.