Several papers describe a modified DNA isolation protocol with the common DNeasy Blood & Tissue Kit, which introduce an additional step of incubation at 70 °C for 10 min after adding the AL buffer and before adding 100% ethanol (e.g. here Article Impact of Phlebotomine Sand Flies on U.S. Military Operation...
).I was wondering why such additional step is needed? Is that a "trick" to increase DNA yield from a source where it is expected to be reasonably low? I have read that the AL buffer contains guanidine hydrochloride, which is helping in denaturing proteins. So, could it be that the incubation at increased temperature further aids this process? Any other ideas as to what this additional step might be doing to the sample?
I would propose to take a look in the protocol/manual in this context, and/or to contact scientific support of the DNeasy Blood & Tissue kit
From my own experience, it helps dissolve a thick, mucus-like white precipitate that sometimes forms when AL buffer is mixed with the enzymatically digested tissue sample in ATL buffer. Heating the mixture for 10 min at 70 °C prior to the addition of absolute ethanol always dissolved most if not all of the precipitate. Sometimes, I ended up with having more than half of the liquid sample become this yucky stuff. If you do not dissolve it, it will most likely clog the column. Been there, done that... 😅 However, if no white precipitate forms in your samples, I reckon you can skip that entire step.
I'm not entirely sure what that precipitate may be (proteins?) but I suppose it is not the same as the one mentioned in the handbook which sometimes floats around in the AL bottle which you should avoid aspirating. In the handbook, they also mention another white precipitate forming after mixing AL and ethanol but personally I have only seen precipitate form when AL, ATL and tissue lysate were mixed (without abs. ethanol).
Thanks for sharing your experience, Marek! I've never seen such precipitates after adding the AL buffer but I guess this might depend on the initial source you are isolating your DNA from. What's interesting though, is that the "10 min at 70 °C" step is not included in any of the official DNeasy Blood & Tissue Kit protocols, so I thought there should be a reason behind introducing it. Your explanation sounds plausible but some of the protocols including this step are "non-destructive" extractions from insects where you leave the specimen intact (except for making several small punctures in the exoskeleton). I'd guess there will be not that much stuff to clog the column after lysing such samples...
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