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Hi
We are using the gDNA protocol 7.1 at a microbiology class, where we grow pure cultures from marine isolates, do 16S amplification and analysis and then sequence some of the interesting isolates with Oxford Nanopore using the rapid sequencing kit. DNA isolation works well, but achieving the required concentration of more than 50 ng/µl is sometimes problematic, depending on the isolate, even with reduced elution buffer volume
What we observe is that the addition of isopropanol after the lysis step causes a white precipitate, which subsequently binds to the beads and if handled carefully can also be washed and eluted. However, it does not dissolve in the elution step in spite of increasing the elution buffer volume. Heating it to 65 C for a couple of minutes will dissolve the precipitate eventually and increase DNA concentration but also decrease the DNA to contamination ratios. Therefore, subsequent use of the DNA for sequencing seems to be rapidly destroying the pores of the nanopore flow cells and results in low coverage of the genome.
As this precipitate does not always form and the concentration and purity is much better in isolates which do not form this precipitate, I wonder if anyone else has a white precipitate forming or if it specific to some of our marine isolates. Removing the precipitate before advancing with further washing and elution will increase the purity but reduce the concentration below the threshold we need for sequencing.
Matti
Hey that seems like a really cool lab, I am looking to implement something similar. I suppose the easiest thing to do is just try skipping the isopropanol step. Now, one thing to note though is that a bunch of white stuff is kind of vague, could it be that there is so much genomic DNA it is precipitating to the point it is visualized? Part of the tricky thing here is even a little bacteria should produce WAY more than 50ng/uL of genomic DNA. How thick of a culture are you using. Another thing you could try is pelleting the precipitate or using a slightly different lysis method, I’ve tried garnet beads for 2 minutes on regular old votrexers and it works OK but not great.
Let me know if you ever figured it out!
Ahoy,
I too have experienced significant issues when trying to isolate DNA from overly contaminant rich sources, massive amounts of white precipitate etc.
My guess was that this has to be some sort of water soluble proteins which will get bound to mag-beads once precipitated. Could you try incorporating a proteinase K step?
Tim
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