Hello, I am working with a strain of Xanthomonas (Xanthomonas campestris pv. vesicatoria) and I want to isolate genomic DNA from this bacteria manually. Can anyone suggest a standard protocol for genomic DNA isolation specific to this Xanthomonas strain ? Thanks in advance
There is no specific for Xcv method for DNA extraction.
You can use any simple method, like this :
Bacteria (log stage of growth) are sub-cultured and grown for 24 hours on LB/YDC agar medium. One loop of culture is suspended in 400 mkl TE buffer (10mM Tris Hydrochloride; 1 mM EDTA). Thoroughly mix it with added 30 mkl of 10% SDS (sodium dodecyl sulphate) and 100 mkl of 5M NaCl. After adding 80 mkl of 10% CTAB (cetyl trimethyl ammonium bromide), incubate it for 15 min at 65oC. Then, 300 mkl of chloroform should be added and mixed thoroughly by vortexing 2 times. Centrifuge it for 15 min at 14,000 rpm, 300 ul of supernatant must be carefully taken to a clean tube and mixed well with 750 ul of 96% ethanol, centrifuged for 10 min at 14 000 rpm. The supernatant must be decanted, the precipitate washed with 200 mkl of 70% ethanol, dried at room temperature for 10-15 minutes, can be kept in 50 mkl of TE at room temperature until complete dissolution of the DNA. DNA concentration is measured by electrophoresis (5 microliters of DNA mixed with stain and applied to 1.5% agarose gel), and diluted to a standard concentration (according to your needs). Original DNA can be stored at – 20oC, and diluted DNA - at +4oC (for short time).
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