How to do effective SPRI beads cleaning

SPRI beads cleaning is one of the most repetitively used step during library preparations and probably the step where most of us lose a lot of precious DNAs.

Losing DNA scared me so much (because it can be observable) that I hesitated a lot before trying to use beads to concentrate genomic DNA, because usual rate of recovery are ~50%.

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Hopefully with some practice and a lot of patience, it is possible to reach 90% recovery. How to lose as little DNA as possible? Here are some guidelines: Continue reading

How my PhD was saved by DTT, or: how to get moderate quantities of clean, unsheared DNA from tricky tissues

I have been trying for months to find a high-throughput solution to DNA extraction from lyophilized (freeze-dried) leaf discs of Helianthus argophyllus, a species known for its intransigent polyphenolics and low DNA yields. And I appear to have found one, thanks to Horne et al. 2004 (here) and Dow Chemical!

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Ph*cking phenolics!

Hi guys,

I have extracted sunflower DNA a dozen times now.  I get one consistent problem a gelatinous film coprecipitating with my DNA (carbohydrates/polysaccharaides).  I also get sharp smelling phenolic compounds.  These occur regardless of DTT/2-BME addition.

I found a paper detailing the use of 2-butoxyethanol (2-BE) to remove strawberry polysaccharides and phenolics.  The researcher’s idea is to perform a two-step precipitation:

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