1202
Fig. 2.
RAPD-PCR assays for the selection of primer concentration using 25 ng DNA of three Lady’s slipper
orchid species; Lanes 1, 4, 7, 11, 14 and 17:
Paphiopedilum thaianum
; Lanes 2, 5, 8, 12, 15 and 18:
P. niveum
;
Lanes 3, 6, 9, 13, 16 and 19:
P. godefroyae
.
Lanes 1-9: primer AA10; Lanes 1-3, 4-6 and 7-9 are 0.5, 1 and 1.5 ôM concentration of primer
respectively.
Lanes 11-19: primer AA11; Lanes 11-13, 14-16 and 17-19 are 0.5, 1.0 and 1.5 ôM concentration of primer
respectively.
Lane 10 and 20 are 1 kb DNA ladder.
Conclusion
The isolated DNA by modification of the microprep method was high purity and compatibility for
RAPD technique. The best result of optimized RAPD-PCR reaction contained 25 ng of template DNA, 1 ôM
primer, 20 mM Tris-HCl, 80 mM KCl, 4 mM of MgCl
2
, 0.5 mM each of dATP, dTTP, dGTP and dCTP and 1 U
of Taq DNA polymerase in 20 ôl of reaction volume. The PCR was programmed for 35 cycles at 93°C for 30 sec,
28°C for 30 sec, 72°C for 45 sec and followed by final extension at 72°C for 5 min. The present optimized
protocol for DNA isolation and RAPD-PCR condition are suitable for further work on genetic diversity and
relationships studies of Lady’s slipper orchids.
Acknowledgments
This work was supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training
grant BRT T253034. The authors wish to thank the heads of Sra Nang Manora Forest Park (Phang-nga province),
Forest Resource Management Office 12 (Krabi province), Tan Bok Kohrane National Park (Krabi province) and
Hat Chao Mai (Trang province) for necessary permits.
