determination of genetic diversity between eggplant and its wild ...

DNA. The products were prepared for gel electrophoresis by adding 2 µl blue juice to
each sample and the gel was 3% agarose 1xTAE. Samples were electrophoresed for at
least 4 hours at 120 mA. For visualizing of DNA bands in the gel, ethidium bromide
was used either by adding it directly to the gel or by staining the gel with an ethidium
bromide solution after electrophoresis. At last step, these gels were viewed using the
AlphaImager Gel Documentation System (Alpha Innotech, San Leandro, CA, USA).
2.2.2.2. SSR Protocol
As a result of gel electrophoresis, primers giving polymorphic bands were
detected. These primer combinations’ forward pairs were extended by adding M13
sequence. M13 sequence was added to the 5’ end of the forward primer whereas the
reverse primer remained same as previously designed (Table 2.7.). These newly
designed forward primers and separate fluorescent M13 primers were synthesized by
Sigma-Proligo (Sigma-Aldrich Company, LTD Irvine, Ayrshire, UK).
The best PCR conditions and the amounts of components in the experiments
were determined after several preliminary experiments. PCR reactions were 20 µl total
for each sample and were composed of 13.15 µl dH2O, 2 µl 10x PCR buffer, 0.4 µl
dNTP, 0.2 µl Taq Polymerase, 0.75 µl of each primer (F and R primers and M13) and 2
µl sample DNA. Sample DNA concentrations were adjusted ~10 ng/µl by dilution with
dH2O. The profile of the PCR was: 94ºC for 5 min.; 94ºC for 30 s., 56ºC for 45 s., 72ºC
for 45 s. for 27 cycles; 94ºC for 30 s., 53ºC for 45 s., 72ºC for 45 s. for eight cycles;
72ºC for 10 min, hold at 4ºC.
Before loading the samples for analysis in the CEQ 8800 Genetic Analysis
System, PCR products were diluted 1:10 with sample loading solution (SLS). For each
sample, 3 µl PCR products were diluted with 27 µl SLS and 0.5 µl size standard-600.
Suitable with the used size standard, Frag 4 method in the system was used. The
profile of the method was: capillary temperature 50ºC, denaturation temperature 90ºC
for 120 sec., injection voltage 2.0kV for 30 sec. and with a separation voltage 4.8 kV for
60.0 min. After definition of the plate and method, system was started to be run.
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