Alright, here's a tricky case. I am trying to extract DNA from mixed marine faunal samples, something that has been done in my group for a long time, and which I have done successfully before as well. Now it's just not working anymore for my last batch of samples, even though they should be very similar to my previous samples, and I'm using the exact same extraction kit (ZymoBiomics DNA Miniprep kit D4300). The yields are much lower than expected, and the Nanodrop spectrophotometer is showing quite a bit of contamination through the 260/230 ratios (see table below). My goal is to do metabarcoding of a couple of marker genes to determine species composition, so I need the DNA extracts to represent their whole communities. When yields are so low, I believe the chance of severe bias increases. I have heard of people getting 200-300 ng/µl from marine sediment, and my samples should in theory contain even more. Nevertheless, what I have gotten used to from earlier extractions are yields around 30-100 ng/µl (all values based on 100 µl). This is something different though. The table below shows the best results out of four extraction attempts. I have double-checked the concentrations with Qubit and they are approximately the same. I should also note that I ordered a totally fresh kit for this extraction, to rule out that something had gone bad in the previous one. But it's not much of a difference. I have also followed Zymo's advice to increase DNA yield: heat up the elution water, let it incubate for longer, run it through the spin column multiple times. It does not significantly affect the results. We preserve our samples in DESS (20% DMSO, 0.25M EDTA, NaCl saturated solution). Zymo suggested that the high salt content could disturb the extraction process. I'm not too sure, since it has worked earlier. But maybe that could be the reason why we are never seeing >200 ng/µl yields? What do you think?

What makes me believe that it's not the samples that have gone bad, is: a) That they would need to get really damn bad before yielding this little DNA, and b) That between every extraction attempt, there is usually one or two samples that spike and show quite ok DNA concentrations (30-60 ng/µl) – but rarely the same samples.

Sample ID Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 7.6 ng/µl 0.152 0.086 1.76 0.71 DNA 50
2 24 ng/µl 0.479 0.249 1.93 1.16 DNA 50
3 21.9 ng/µl 0.439 0.238 1.84 1.15 DNA 50
4 18.6 ng/µl 0.371 0.190 1.95 1.29 DNA 50
5 18.5 ng/µl 0.371 0.200 1.85 1.44 DNA 50
6 6.9 ng/µl 0.138 0.069 2.00 0.70 DNA 50
7 7.9 ng/µl 0.157 0.092 1.72 0.78 DNA 50
8 35.2 ng/µl 0.703 0.363 1.94 1.67 DNA 50
9 9.6 ng/µl 0.192 0.099 1.94 0.85 DNA 50
extraction_blank 1.6 ng/µl 0.031 0.006 4.84 0.19 DNA 50
collection_blank 3.9 ng/µl 0.078 0.036 2.19 0.42 DNA 50
  • $\begingroup$ I don't see that this question is significantly different to your previous question, which is essentially the same the question as here. Either way, you will need a bit more detail on the samples - how are they stored, how much input, what is in the samples (tissue? soil?), what has changed between previous extractions and now, have you gone back to old (known working) samples and tried re-extracting? $\endgroup$
    – bob1
    Commented Jan 9 at 20:30
  • $\begingroup$ Does this answer your question? Can contaminants in the DNA extract disturb the sequencing? $\endgroup$
    – bob1
    Commented Jan 9 at 20:30
  • $\begingroup$ I'd suggest trying an alternate sample prep method that is a little easier to introspect than the kit and is optimized for difficult samples. For example this chapter describes a "difficult sample" extraction method that works on soil or fungi, which are notoriously hard to get DNA out of. $\endgroup$ Commented Jan 9 at 21:21
  • $\begingroup$ Bob: The samples are mixed marine faunal samples. That is, a mixture of anything larger than 40 µm that lives on a settlement plate in the sea, or swims near it at the time of retrieval. Directly upon retrieval, samples are mortarted thoroughly and preserved in DESS as described above, then stored in -20℃. What changed between the previous extractions and the last one is that I got a fresh ZymoBiomics DNA Miniprep kit for the last extraction. For the previous extractions I played around a bit with the tips that I got from Zymo, but the point is that it didn't make any significant difference. $\endgroup$
    – Joel
    Commented Jan 10 at 0:14
  • $\begingroup$ Today I also tried with the Qiagen DNeasy PowerSoil Pro kit, but the results were practically the same, or possibly even slightly worse. So I will go back to the Zymo kit. Maybe redo some old samples that I know have been working before... $\endgroup$
    – Joel
    Commented Jan 10 at 0:23


You must log in to answer this question.

Browse other questions tagged .