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Materials and methods
Plant materials and their genomic DNA extraction
Paphiopedilum thaianum
,
P. niveum
and
P. godefroyae
were collected from southern Thailand.
The genomic DNA was extracted from young leaves by a modification of the microprep method (Fulton
et al.
,
1995). Approximately two hundred milligrams fresh leaf tissue was ground to powder in liquid nitrogen using
a mortar and pestle and then transferred to eppendorf tubes. The tissue powder was homogenized in 700 ôl of
extraction buffer (100 mM Tris-HCl pH 8, 50 mM EDTA pH 8, 500 mM NaCl, 1.25% SDS (w/v), 8.3 mM NaOH,
0.38 g sodium bisulfite per 100 ml) and incubated in 65°C water bath for 120 min. The mixture was extracted once
with 650 ôl of chloroform: isoamyl alcohol (24:1,v/v) and centrifuged at 13,000 rpm for 10 min. The clear
supernatant was transferred to a new tube and then added 65 ôl of 10% cetyltrimethylammonium bromide
(CTAB) solution in 0.7 M NaCl and 600 ôl of phenol: chloroform: isoamyl alcohol (25: 24 :1,v/v). The mixture
was shaked for 10 min and centrifuged at 13,000 rpm within 10 min. The clear supernatant was removed into
a new tube and extracted again with chloroform: isoamyl alcohol. The DNA was precipitated from the supernatant
by addition of a volume of cold isopropanol and centrifuged at 13,000 rpm for 5 min. The DNA pellet was washed
twice with 70% ethanol, air dried at room temperature and resuspended in 50 ôl of TE buffer
in 65 °C water bath
for 5 min. DNA concentrations were determined by measuring the absorbance at 260 nm. The DNA quality was
assessed by examination on 0.8% (w/v) agarose gel stained with ethidium bromide (0.5 ôg/ml).
PCR optimization
PCR reactions were performed in 20 ôl reaction volume containing 10 ôl
Prime Taq
Premix (2x) (Genet
Bio, Korea) with varying template DNA concentration (20, 25, 50 and 100 ng) and primer concentration (0.5, 1.0
and 1.5 mM). Four primers were used in this study as follows: OPD07, AA10, AA11 and AA12.
The thermocycler was programmed for an initial denaturation step of 4 min at 93°C, followed by 35 cycles of 30
sec at 93°C (denaturing), 30 sec at 28°C (annealing) and 45 sec 72°C (extension). Additional final extension at
72°C for 5 min and a hold temperature of 10°C at the end. PCR products were electrophoresed on 1.5% (w/v)
agarose gels, in 1X TBE Buffer at 60 Volts for 60 min and then stained with ethidium bromide (0.5 ôg/ml).
Gels with amplification fragments were visualized and photographed under UV light.
Results and discussion
DNA extraction was improved by modified microprep protocol (Fulton
et al.
, 1995). The result was high
quality, low-polysaccharide genomic DNA from three different Lady’s slipper orchid species. The yield of pure
DNA ranged from 80-500 ng per microliter. The addition of high concentration CTAB (10% w/v) in 0.7 M
sodium chloride helped removal the remaining polysaccharides in the extracted DNA. The emulsification-washes
with phenol: chloroform: isoamyl alcohol efficiently removed high protein and the CTAB-polysaccharides
