[Kou maru]

Nice to meet you. I am a university student in Japan. I am very struggling to extract and purify the DNA from soil. My professor is so old thinker and would like to make individual method, moreover it is cheaper. However, this is fool thinking and he does not absolutely buy general using kits. Therefore, I am struggling now. So I have a question to you. As you can see the subject, could this Magnetic Beads method apply the extraction and purification from soil?
Now, my using method is written below.

1. stainless steel beads 0.5 g, sample soil 0.5 g, extraction solution 1 mL, 20% SDS 0.2 mL, shaking tubes contain and shake in about 4000 rpm, 5 min.
2. after that, centrifuge in 15000 rpm, and transfer the supernatant 600 µL to micro tube and add the chloroform/isoamilalcohol same amount, resuspend and centrifuge.
3. transfer supernatant to new tube and add the 2-propanol, then centrifuge.
4. decant the supernatant and wash in 70% ethanol.
5. in the end, dry and resolve in TE solution or distilled milliQ water.

1-5 is extraction DNA way, but sample DNA solution contains much humus and so on. So, these are not clear and mainly colored blown or like black. therefore I make mistake and cannot apply PCR and Real-time PCR. purification method is gel cutting. But that is not efficient and lack of precision. Please help me.

I could not find the “soil” character but “environmental” exists. So could you apply this method to extract and purify the DNA from soil? I asked. Thank you in advance.

I am looking forward to hearing from you.

[Phil Oberacker]

Hi Kou,

I can only support trying to find individual do-it-yourself methods as that’s how you really understand what you’re doing 😀 otherwise you’ll become a kit-scientist 😉

I would indeed recommend the environmental TNA protocol (#6.7) as a reference. Dissolve your soil sample in e.g. water or TRIS-buffer (in the protocol we went with 50 ml lake water) and filter it first with a common coffee or paper filter (to get rid of the large humus- and dirt-particles) and maybe a second time with a tighter filter, depending on what exactly you want to find. Just don’t go with filter pores that are tight enough to catch the bacteria, fungi or general species you want to detect in your samples. Afterward, you can just follow the protocol, starting with Step 2 (centrifugation).

While checking the protocol I realized that it just says coated magnetic beads. We use silica-coated magnetic nanoparticles for this. You can easily make them yourself by following BOMB protocols #1.1 and #2.1 or this description, in which we went far more into detail: https://bio-protocol.org/e3394. Alternatively, you could buy them commercially, of course.

In case you want to isolate gDNA only, you can add RNase A to the TE buffer as described in protocol #7.1. If it’s RNA you’re looking for, I would switch protocols after Step 10 (drying after washing) to protocol #8.2 Step 13 (DNase I reaction mix) and go on like described there. And keep in mind that these protocols were designed for Deepwell plates, meaning for the DNase digest you can right away go to 37 °C at 1300 rpm or so if you are working with 1.5 ml tubes or so (it’s just important that the beads are dissolved and not clumped as a pellet on the ground).

Hope I could help a bit 🙂 If you have any more questions, don’t hesitate to ask!

Cheers
Phil

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