Application Note
Detection And Avoidance Of Polysaccharides In Plant Nucleic Acid Extractions
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•Application Note: Detection And Avoidance Of Polysaccharides In Plant Nucleic Acid Extractions
By Thermo Scientific NanoDrop Products
Spectrophotometric measurement of nucleic acids is common, both in order to quantify the DNA or RNA, and also to assess purity. By measuring the absorbance at 260 nm, it is possible to calculate nucleic acid concentration. Absorbance at other wavelengths, particularly 280 and 230 nm, may be used as a measure of purity of the sample by calculating the ratio of the absorbances at these wavelengths to the absorbance at 260 nm. Because proteins have an absorbance maximum at 280 nm, the A260/A280 ratio may be used to assess protein contamination: a ratio of less than 1.6 is commonly considered poor, while ratios above 1.8 are considered good. Similarly, because many polysaccharides absorb at 230 nm, a A260/A230 ratio may be used for their detection: ratios of 1.6 or less are generally considered poor, while ratios above 1.9 indicate very little polysaccharide contamination.
The A260/A230 ratio is of special importance to plant molecular biologists because plant samples typically have relatively high polysaccharide contents. In addition to cell wall material and sugar or starch content, plant cells contain a vast (and often species specific) array of polysaccharides. Removal of these has led to many laboratories developing species-specific protocols. Generally, plant nucleic extractions begin with tissue homogenization, either in buffer, or using liquid nitrogen before the addition of buffer. The extraction buffer used is a key component of the procedure, with each ingredient playing a specific role. The addition of sodium chloride to the buffer helps to prevent polysaccharides from precipitating following the addition of alcohol. Polysaccharides are therefore discarded in the supernatant following nucleic acid precipitation and centrifugation, resulting in DNA pellets with low polysaccharide contamination.
Click Here To Download:•Application Note: Detection And Avoidance Of Polysaccharides In Plant Nucleic Acid Extractions
