National Academy of Agricultural Sciences (NAAS)
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PRINT ISSN : 2319-7692
Online ISSN : 2319-7706 Issues : 12 per year Publisher : Excellent Publishers Email : editorijcmas@gmail.com / submit@ijcmas.com Editor-in-chief: Dr.M.Prakash Index Copernicus ICV 2018: 95.39 NAAS RATING 2020: 5.38 |
A simple and easy protocol for isolation of DNA from major plant pathogen Alternaria spp has been developed in which DNA extracted using CTAB extraction method from fresh mycelium of fungi crushed with sterile sand particles and liquid nitrogen. The DNA was precipitated first with equal volume of phenol-chloroform-isoamylalcohol (25:24:1) and further with equal volume of chloroform-isoamylalcohol (24:1). Repeated centrifugation was done at 10000 g for 10 minutes at room temperature. DNA was pooled by incubating the supernatant with pre-chilled isopropanol at -20°C for 1 hour. After that the samples were centrifuged for 15 minutes at 13000 g to pellet the DNA. The DNA pellet was washed with 200 µL of 70% ethanol, air dried and dissolved in 200 µL of TE buffer, thereafter 5 µL of RNase was added to each DNA Samples, mixed and incubated at 37°C for 1 hour. After RNase treatment, DNA samples were precipitated with chloroform-isoamylalcohol (24:1) again incubated with pre-chilled isopropanol and then DNA pellet which was finally dissolved in 20 µL of TE buffer and stored at -20°C till further use. Inter-simple sequence repeats (ISSRs) amplification reaction was carried out using 20 µL of reaction volume containing 2 µL of DNA.
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