*4.4. DNA Isolation and PCR Amplification*

A rice genomic DNA extraction, with modification, was adopted for minipreparation [45]. Approximately 0.05 g of fresh leaf tissue from 6- to 8-week-old seedlings was homogenized with 300 μL extraction buffer (100 mM Tis-HCl, pH 9.0; 40 mM EDTA-2Na, pH 8.0; 1.67% SDS) at 30 1/s for 2 min by use of TissueLyser (Qiagen Retsch GmbH, Haan, Germany). A total of 150 μL benzyl chloride was added to the homogenized tissue and vortexed. After incubation in a 50 ◦C water bath for 15 min, 150 μL of 3 M sodium acetate (pH 5.2) was added. Supernatants were saved after centrifugation at 15,000 rpm for 15 min at 4 ◦C, and 300 μL of ice-cold isopropanol was added to precipitate DNA. After centrifugation at 15,000 rpm for 10 min, DNA pellets were saved and washed with 70% ethanol, air-dried and dissolved in 50 μL TE buffer.

A 10 μL PCR reaction containing 20 ng genomic DNA, 0.2 μM forward and reverse primers, 5 μL Multiplex PCR Master Mix (QIAGEN, Inc., Valencia, CA), and 1 μL Q-Solution (QIAGEN, Inc., Valencia, CA) was performed by use of a thermocycler (GeneAmp PCR System 9700, PerkinElmer Corp., Norwalk, CT, USA) at 95 ◦C for 15 min for 1 cycle; 94 ◦C for 30 s, 57 ◦C for 2 min, 72 ◦C for 2 min for 30 cycles; and 60 ◦C for 30 min for 1 cycle. Following PCR, 2 μL of amplified DNA products was separated by 6% polyacrylamide gel (PAGE) in 0.5 × TBE at 100 v (Dual Triple-Wide Mini-Vertical System, C. B. S. Scientific, CA, USA) for 60 min.
