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13. SUPPLEMENTARY MATERIALS
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ANNEX 13.6. AMPLIFICATION OF CHLOROPLAST DNA (cpDNA)
Total DNA of individual plants was used in PCR reaction. The non-coding region of
chloroplast genome bounded by psbC (psII 44 KD protein) and trnS [tRNA-Ser(UGA)] conservative
coding regions was amplified by the pair of “universal primers” with following sequences:
• psbC-trnS-F 5’GGT CGT GAC CAA GAA ACC AC3’
• psbC-trnS-R 5’GGT TCG AAT CCC TCT CTC TC3’
At the first step, the annealing temperature was optimized using PTC200 gradient
thermal cycler (BioRad). Each PCR reaction was performed in a total volume 20 µl. Concentrations
of reagents and thermal conditions are shown in Table 13.6.1. PCR products
(5 μl) were loaded on 1.5% (w/v) agarose gels containing 0.5 µg/ml ethidium bromide and
separated in 1 x TBE buffer (Tris-Borate-EDTA; Sambrook et al. 1989) at 100 V constant
power. PCR products were digested with three restriction enzymes as follow: DraI, EcoRI
and HaeIII. Each reaction mixture of 20 µl contained 5 µl of a PCR product and 3 U of restriction
enzyme. Restriction fragments were separated on 2% agarose gels, visualized under UV
light (312 nm), photographed and stored as .jpg files. Each restriction fragment was scored
as present (1) and absent (0).