DNA was extracted using modified CTAB method [25 ] and was quantified using a spectrophotometer. Thirty SSR markers which were polymorphic between two genotypes that a showed contrasting response to alkalinity stress, along with additional 38 arbitrary SSR markers were used for screening 285 genotypes. These markers were selected based on earlier lentil reports published [16 (link), 26 (link), 27 (link)].
PCR amplifications were performed in 10μl reaction volume, consisting of 1 X PCR buffer, 1.5 mM MgCl2 and 0.5 μM primers each of forward and reverse, 1 mM dNTP, 0.5 U Taq DNA polymerase and 50 ng template DNA. PCR cycling conditions were as follows: Pre-denaturation at 94°C for 3 min followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, elongation at 72°C for 1 min with a final extension at 72°C for 10 min. PCR amplified products were separated on 3% ultra high resolution agarose gels and documented using Vilber Lourmat Gel Documentation System.
PCR amplifications were performed in 10μl reaction volume, consisting of 1 X PCR buffer, 1.5 mM MgCl2 and 0.5 μM primers each of forward and reverse, 1 mM dNTP, 0.5 U Taq DNA polymerase and 50 ng template DNA. PCR cycling conditions were as follows: Pre-denaturation at 94°C for 3 min followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, elongation at 72°C for 1 min with a final extension at 72°C for 10 min. PCR amplified products were separated on 3% ultra high resolution agarose gels and documented using Vilber Lourmat Gel Documentation System.
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