Hi all,
I’m working on a metagenomics project focused on extracting the whole microbiome from seeds. My protocol for the DNA extraction involves soaking seeds in 0.85% saline for 4 hours, then shaking them at 150 rpm for 10 minutes at room temp. I then proceed with DNA extraction using the Qiagen DNeasy PowerSoil Pro Kit, following the standard manufacturer protocol.
In previous experiments, this workflow gave me very high DNA yields. However, in my recent extractions, even after increasing the seed amount, I’m consistently getting low concentrations (around 2–3 ng/µl using the Qubit dsDNA HS kit).
Has anyone else experienced a similar drop in yield despite following the same protocol? I’d really appreciate any advice or troubleshooting tips.
Thanks in advance for your help!
Why are you using the kit for soil and not the kit for plants? The Qiashredder columns are designed for plant cells & are in addition to grinding/bead beating.
What type of seeds? If they have a high oil content, that can make it challenging to get DNA. For Arabidiopsis seeds, I used a protocol with phenol and chloroform. Grinding really well is also needed. Do you have a bead-beater? Or tiny pestles to grind in 1.7 ml tubes?
For seeds with a tough outer covering, like rice, remove the hull/husk/awn.
Good luck!
I agree on the grinding well aspect, it's essential and not always easy. You can add a bit of sand for grinding with a mortar/pestle. It will pellet away with any cell debris and won't carry through. I've used this approach for barley seeds and corn kernels.
Hi Mozhde Hamidizade , there are many reasons resulted in low DNA yields. Poor quality or degraded DNA is one of the reasons. In this case, with no extraction steps which can contribute to DNA loseses, native/orgininal DNA from supernatant (after physical fragmentation using a pestle) can be a DIRECT source for DNA profiling. Please see our method published.
https://doi.org/10.1111/1556-4029.15033
Hope it helps,
Good luck and regards
Thien
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