1201
complex (Harini
et al.
, 2008). The DNA quality has a great effect on the generation and resolution of amplified
products because the amplification process requires such small amounts of template DNA. Extraction procedures
emphasize purity rather than quantity (Weeden
et al.,
1992). The DNA quality from this study was acceptable to
produced scorable and clear amplicons in all samples (Fig. 1).
Fig. 1.
RAPD-PCR assays for the selection of DNA concentration using primer OPD-07 and DNA of three
Lady’s slipper orchid species; Lanes 1, 4, 7 and 10:
Paphiopedilum thaianum
; Lanes 2, 5, 8 and 11:
P. niveum
;
Lanes 3, 6, 9 and 12:
P. godefroyae
; Lane marked M are 100 bp DNA ladder; Lanes 1-3, 4-6, 7-9 and 10-12 are
20, 25, 50 and 100 ng concentration of DNA respectively.
The little change of any ingredient concentration would affect RAPD-PCR products drastically (Diakou
and Dovas, 2001; Fraga
et al
., 2002). Thus,
Prime Taq
Premix was control parameters in the present optimization
system. It contains 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 Taq DNA polymerase. The variation in template DNA and primer concentration were investigated
to obtain the best RAPD profile. Amplification was performed using primer OPD-07, AA10, AA11, AA12 with
range of 20-100 of purified DNA as template. The examination was repeated two times. The best banding patterns
were observed at 25 ng DNA concentration. Higher concentration of DNA showed the worst amplification (Fig.
1). The concentration of primer had an influence on the amplification as well. Primer concentration in range of
0.5-1.5 ôM were examined with all primers. The good result was exhibited with 1.0-1.5 primer concentration.
However, three primers revealed different clarity of RAPD profiles (Fig 2).
