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Help with plant DNA contaminants and troubleshooting suggestions

DNA plant DNA contaminants proteins polysaccharides

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#1 paula1



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Posted 04 May 2013 - 10:46 AM


I've been having problems extracting DNA from silica gel dried leaves from a plant known to have a serious polysaccharide contamination issue. It also has high quantities of tannins and polyphenols. The resulting DNA is very viscous and the nanodrop profile is catastrophic. I get samples with a 260/280 ratio of 1.3 and a 260/230 of 0.14 (seriously). I use a protocol for obtaining high quality DNA from plants with high quantities of polysaccharides (Russell at al 2010), with sorbitol buffer.

What I want to know is this: do you think I'd get better results by doubling the concentration of sorbitol in my buffer? Also, given the low 260/280, would you advise me to add proteinase K to the buffer as well? The protocol already contains a phase in which the samples are bathed in CIA for 30 minutes, so I think I shouldn't be getting such bad 260/280 ratios. Maybe increasing the CIA bath time instead of adding proteinase K? I don't know, I'm confused. Any ideas on why this is happening and what could solve my polysaccharide + protein problem?

Thanks a lot! =)

PS: if it helps, I can add the nanodrop results for more information.

Also tagged with one or more of these keywords: DNA, plant DNA, contaminants, proteins, polysaccharides

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