I’m trying to do cleanup reactions for my PCR’s with the protocol. When I go with a ratio of 2, the protocol works fine with any okayish yield of about 60-70% (10 uL PCR product, 10 uL Beads, 5 uL water, 50 uL of binding buffer).

However when I lower the ratio the yield directly drops to ~ 10%. I assume that it is due to the pH of the…Read more

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…Read more

I’m trying to modify some of your protocols to more closely fit my needs (soil, feeces, whole specimen and so on). I succeeed in doing so for extractions from homogenized insects (TNES + Prot K. Lysis, followed by your cleanup protocol). However I’m struggling with some of the protocols especially when it comes to TNA. Would you mind…Read more

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.

I was wondering why it is 2 EtOH washes for the TNA protocols followed by 5-10 minutes of drying and 4 (or 2×4) EtOH washes followed by 30 minutes of drying.

What am I missing here?

best Dominik