FastDigest Eco52I

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	FastDigest® Restriction Enzymes (EagI, EcoRI, FspI, and HindIII) were used according to manufacturer instructions to cleave genomic DNA of the plant materials at specific target nucleotide sequences into DNA fragments of different size. The extracted DNA was subjected to RFLP analysis (Parani et al., 1997). Briefly, the reaction mixture was prepared by adding 10 mL extracted DNA, 15 mL 2X assay buffer, 10 mL bovine serum albumin (BSA), and 3 mL of each restriction enzyme. The vials were incubated at 37°C for 1 h for complete digestion. The restriction enzyme-digested products were visualized through silver staining of the polyacrylamide gel.

Protocol tips

FastDigest® Restriction Enzymes (EagI, EcoRI, FspI, and HindIII) were used according to manufacturer instructions to cleave genomic DNA of the plant materials at specific target nucleotide sequences into DNA fragments of different size. The extracted DNA was subjected to RFLP analysis (Parani et al., 1997). Briefly, the reaction mixture was prepared by adding 10 mL extracted DNA, 15 mL 2X assay buffer, 10 mL bovine serum albumin (BSA), and 3 mL of each restriction enzyme. The vials were incubated at 37°C for 1 h for complete digestion. The restriction enzyme-digested products were visualized through silver staining of the polyacrylamide gel.

Publication protocol

"FastDigest® Restriction Enzymes (EagI, EcoRI, FspI, and HindIII) were used according to manufacturer instructions to cleave genomic DNA of the plant materials at specific
target nucleotide sequences into DNA fragments of different size. The extracted DNA was
subjected to RFLP analysis (Parani et al., 1997). Briefly, the reaction mixture was prepared
by adding 10 mL extracted DNA, 15 mL 2X assay buffer, 10 mL bovine serum albumin (BSA),
and 3 mL of each restriction enzyme. The vials were incubated at 37°C for 1 h for complete
digestion. The restriction enzyme-digested products were visualized through silver staining of
the polyacrylamide gel. The gels were fixed in 50 mL fixing solution (diluted five times with
30.4 mL double-distilled water and 9.6 mL ethanol) for 30 min and silver-impregnated (with
1X staining solution) for another 30 min. Gels were then washed in double-distilled water for
1 to 2 min. After removing the staining solution, the gels were kept in the developing solution
in the dark for 10 min. When the bands were dark enough, the developing solution was poured
out, and the stopping and preserving solution was immediately added. At that time, photographs were taken using a digital system and analyzed with computer software."

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