Effective DNA Extraction Protocol for Kenaf
Journal Title: International Journal of Farming and Allied Sciences - Year 2014, Vol 3, Issue 1
Abstract
Polymerase chain reaction (PCR) has a wide range of applications in molecular biological research. However, the quantity and quality of template DNA have a significant consequence on the reproducibility of PCR results. Kenaf (Hibiscus cannabinus L.) contains high amount of secondary metabolites and polyphenolic compounds, which cause difficulties in the extraction of genomic DNA. When extraction of DNA using three commercial extraction kits failed, CTAB DNA extraction protocol was adopted. This paper describes the steps to optimize the modified Cetyl-trimethyl-ammoniumbromide (CTAB) method for isolation of high quality DNA from plants containing a large amount of mucilage. Concentrations of CTAB, polyvinylpyrrolidone (PVP), -mercaptoethanol ( -ME) and chlorophorm-isoamyl alcohol and duration of incubation at oC were optimized. Minimal presence of contaminating metabolites was confirmed by the ratio of spectrophotometric absorbance of the sample at nm to that of nm (A /A ). Results of this research revealed that extraction buffer containing CTAB, mM -ME, without PVP, with -minute incubation at oC and single application of chlorophorm-isoamyl alcohol mixture was the best treatment. The appropriate concentration of -ME was found to be an important factor in the preparation of extraction buffer for kenaf genomic DNA.
Authors and Affiliations
Rahmatollah Behmaram, Ghizan Saleh, Majid Foroughi, Zahra Noori, Jothi Malar Panandam and Jalaluddin Harun
Keywords
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