DNA isolation from plants is traditionally done using hexadecyltrimethylammonium bromide (CTAB) in the extraction buffer, which binds to polysaccharides and makes them insoluble. CTAB reduces polysaccharide contamination that can cause issues with DNA in downstream applications. Also an issue is polyphenol oxidase that polymerizes monophenols following homogenization. The resulting polyphenols, like polysaccharides, can also hinder downstream manipulations of DNA. A good homogenization buffer will include additives such as PVP (polyvinyl pyrrolidone) to remove polyphenols. CTAB extraction buffers should eliminate these issues, making the isolation of high quality DNA considerably easier.
This study makes a comparison between three different CTAB buffers. These included a homegrown CTAB buffer (2% CTAB, 1% PVP, 20 mM EDTA, 100 mM Tris HCl, 1.4 M NaCl), a commercial CTAB formula (G-Bioscience, cat# 786-565), and the OPS Diagnostics CTAB Extraction Buffer (CEB 500-02, CEB 125-01). The buffers were used to isolate DNA from two week old corn plants.
CTAB Extraction Buffer (OPS Diagnostics) yielded a higher concentration of DNA followed by the Commercial Buffer, and then the homegrown buffer. When considering purity, the CTAB Extraction Buffer performed better then commercial and Homegrown buffers for plant DNA extraction (Table 1).
|CTAB Extraction Buffer||19,141||1.94||1.84|
The DNA samples were scanned using a DeNovix spectrophotomter. The resulting scans are illustrated in Fig 1.
Figure 1. Scans of the DNA preparations from corn using different CTAB buffers. The G-Bioscience buffer is the black line, the Homegrown is the green line, and the CTAB Extraction Buffer is shown in blue.The result from this comparison shows that although CTAB and other additives are important for DNA isolation, other aspects of the buffer formulation are critical. The OPS Diagnostics CTAB Extraction Buffer was analyzed and adjusted to provide higher DNA yields and purity.