[Dominik]

Hi Tim,

thank you for your prompt and detailed answer, yet there remain some uncertainties, which I hope can be resolved. As far as I’m concerned, after whichever lysis, the binding of the DNA/RNA/TNA to the silica (beads / column) is the critical step. After I got my NA to bind to the silica I’m somewhat safe. Therefore I’m trying to figure out the main drivers of the binding:

1. Salt concentration
2. Ethanol / Isopropanol concentration
3. pH?

1. I looked through the protocols and found different final concentrations of salt in the binding procedure. For 4.1 it ranges from 2M – 0.6M while at 0.6M we loose some of the short fragments, which is intended in this case. In protocol 6.7 and 8.2 the binding happens at 1.3M – 1.6M of GITC, for the RNA binding after the DNase treatment it’s 0.8M. I’d conclude that the concentration is not that important as long as it is high enough (probably above 0.8 to bind the full size range), am I right with that?

2. Ethanol / Isopropanol seems to be clear. EtOH above 75%, Isopropanol above 35%. What still confuses me is, that in protocol 4.1 you mix 1 volume of samples with 2 volumes of binding buffer, so you get a final concentration of ~60% EtOH, which should exclude smaller fragments. I’m using this protocol for DNA extractions from tissue, will I loose any of the DNA by doing so? Getting great results so far.

3. pH: This is where I’m most puzzled. You wrote that the binding happens at slightly basic pH, which is certainly not true for the 4.1 and the RNA binding step in protocol 8.2 (which both have a pH below 7). Is there any explanation for this or is pH not the main driving factor? In most spin-column kit (MN, Quiagen) it is even advised to keep the pH below 6.5 indicated by the yellow color of the binding buffer (which I’m guessing is phenol-red?)

Final question:

The reason I’m asking is that I’m trying to modify some of the protocols. For some samples it is easier for me the go with a TNES/Prot. K lysis and bind the DNA from the lysate to the beads with your protocol 4.1. However this does not work well for soil and water samples, I’m getting some nasty inhibitors also indicated by a brown color of the finale DNA elution. When I’m trying to lyse this samples with GITC it gets even worse: The lysate somehow takes away the magnetic properties of the beads (my guess is, that something is co-precipitated and also binds to the beads as well). I found some protocols to remove inhibitors (some proteins, humic substances) from lysates and want to try these. However this will change the salt concentrations in the lysate if I’m doing the GITC lysis. For inhibitor removal, I’m going to use the Flocculant Solution from this protocol (https://www.protocols.io/view/mu-dna-a-modular-universal-dna-extraction-method-a-qn9dvh6/guidelines). Since we are mainly interested in DNA I think I’m fine doing this with TNES lysed samples, but will need to adjust for the GITC lysis if I also need RNA.

Any advice on that or some more insight in how you came up with the protocols?

best Dominik

[Dominik]

Hi Tim,

thanks for the quick reply. For DNA Extraction I usually use 2 washes of 80% EtOH and did not run into trouble with my PCR’s so I guess this is sufficient. For the TNA extraction I will go with 4 then, I will automatize the whole protocol anyways, then there is basically no difference between 2 and 4 washes except maybe a minute longer runtime.

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