Dneasy miniprep kit

Total genomic DNA was extracted from 100 mg fresh leaves of donor and regenerant plants obtained via androgenesis (Experiments 1 and 2) using the DNeasy MiniPrep kit (Qiagen). DNA quantity was evaluated spectrophotometrically, and DNA integrity and purity were verified electrophoretically.

The methylation state of cytosines within genomic DNA was analyzed using a cytosine extension method [59 (link)]. Cellular genomic DNA was extracted from logarithmically growing CEM-C1-15, CEM-IV B9, RPMI 8226, RPMI-II E6, or Molt-4 cells treated with DMSO vehicle or 100 nM AZA for 48 hours using a Qiagen DNeasy mini-prep kit (Qiagen, Valencia, CA). Residual RNA contamination was eliminated by digestion with 400 μg of RNase A. Purified DNA was quantified and 2 μg was digested overnight at 37°C using 8 units of the methylation-sensitive restriction enzyme Hpa II (New England Biolabs, Ipswich, MA). Extension was performed by combining 0.25 μg of digested DNA in the presence of ³H deoxycytidine triphosphate (dCTP) (53.0 Ci/mmol, Perkin Elmer, Waltham, MA), 0.25 units of TAQ polymerase (New England Biolabs), and buffer supplied with the enzyme for 1 hour at 56°C. Samples were subsequently placed on ice and duplicate 10 μl aliquots were bound onto Whatman DE-81 ion exchange filter papers (Whatman Inc., Florham Park, NJ) using a vacuum manifold. Filters were washed 3 times with 0.5 ml room temperature PBS, air dried, submerged in 3 mls of Scintiverse scintillation fluid (Fisher Scientific, Fair Lawn, NJ), vigorously vortex mixed for 15 seconds, allowed to recover for 1 hour, and evaluated using a Beckman scintillation counter (Beckman Coulter).