A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

Abstract

Extraction of high-quality DNA from Gossypium (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10-15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation. •Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively.•Ribonuclease A was added before chloroform extraction.•A simple, rapid and inexpensive DNA extraction method is described.

Keywords: CTAB; CTAB, cetyltrimethylammonium bromide; DNA extraction; Gossypium; PVP-10, polyvinylpyrrolidone; Polyphenols; Polysaccharides; Without liquid-nitrogen.

Graphical abstract

Fig. 1

PCR, restriction and electrophoretic analyses of the isolated DNA from the seed and leaf tissues of cotton. A. 300 ng of undigested DNA and 10 μg of Hind III digested DNA separated on 1% agarose gel. Lane M: λ-Hind III ladder (Fermentas, USA); lane 1 and 3: undigested leaf and seed; lane 2 and 4: Hind III digested leaf and seed. B. PCR amplification of the CP4-EPSPS gene (111 bp). Lane M: 50 bp ladder; lane 1: positive control (Recombinant plasmid DNA harboring the CP4-EPSPS gene sequence); lane 2: negative control (without any template DNA); lane 3 and 4: amplified sequence from leaf and seed DNA.