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Old 05-17-2018, 03:41 PM   #1
anjama
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Location: Florida

Join Date: Mar 2016
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Default CTAB alternative for carboxyl mag bead gDNA extraction

Last summer I developed a protocol for our lab to extract gDNA from tissue samples using the carboxyl coated magnetic beads used in SPRI. It has worked really well with a lot of advantages including increased throughput, reduced cost, and very clean samples. Cell lysis is performed in a Tris, EDTA, NaCl, SDS solution with Pro-K, which is then mixed directly with the PEG and NaCl bead solution.

Now I'm trying to modify it for another project to extract DNA from epithelial cells in fecal samples of herbivores. As is, the protocol does appear to successfully extract DNA, but the quality is terrible and doesn't seem to work in a PCR. Presumably, there are polysaccharides and phenols carrying over in the process from the plant material in the fecal sample, which could explain the poor quality scores and failed PCRs.

My next step was to introduce an extra step after the intial bead extraction where I place the beads in a 2% CTAB lysis (with Polyvinylpyrrolidone, aka PVP) solution to try and remove the saccharides and phenols. I did this step separately from the initial lysis because it's my understanding that the SDS (anionic) and CTAB (cationic) could precipitate out with each other. However, I'm finding that mixing the beads with the CTAB is causing the beads to clump up and float. My guess is that the relatively negative charge of the beads is causing them to form micelles with the cationic CTAB.

Are there any suggestions to keep this from happening? Or an alternative to CTAB for removing polysaccharides that is bead friendly?
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Old 05-17-2018, 07:56 PM   #2
austinso
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Location: Bay area

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Default

Yes...that would be expected...can't really avoid it either...it depends on the coating on the bead (they have slightly different versions).

So one thing you could do is pellet the nucleic acids (well all negatively charged polymers) in the CTAB solution (I forgot what the w/v concentration should be to do that...and don't know if PVP offsets that) and remove the soup, then resuspend in SDS and then do the bead clean up with that resuspended material.

But...hard to know without more details...
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