NucleoSpin Experiment

Owned by Shannon Harris
Oct 21, 2021
3 min read

We will be using 10 samples from Josh’s Populus project in duplicate to test the Macherey-Nagel Genomic DNA from Soil protocol/kit.

Extraction Protocol

Before starting the preparation, check Lysis Buffer SL1 or SL2 for precipitated SDS. Dissolve any precipitate by incubating the buffer at 30–40 °C for 10 min and shaking the bottle every 2 min.

See section 2.5 and 2.6 for more information on the amount of starting material and the choice of lysis buffer.

  1. Transfer 250–500 mg fresh sample material to a MN Bead Tube containing the ceramic beads. Important: Do not fill the tube higher than the 1 mL mark.

  2. Add 700 µL Buffer SL1 or Buffer SL2. Note for very dry material: If the sample material soaks up too much lysis buffer, fill the MN Bead Tube up to the 1.5 mL mark with fresh lysis buffer. Note for very wet material: Remove excess liquid before addition of lysis buffer, if necessary after spinning down the sample.

  3. Add 150 µL Enhancer SX and close the cap. Note: Enhancer SX ensures the highest possible DNA yield. It can, however, also promote the release of humic acids. See section 2.6 on how to lower the volume or omit the buffer entirely in order to increase DNA purity.

  4. Load the MN Bead Tubes onto the mixer mill and run at 5m/s for 30sec. Check lysate and repeat up to 2min.

  5. Centrifuge for 2 min at 11,000 x g to eliminate the foam caused by the detergent. Note: The clear supernatant can be transferred to a new collection tube (not provided) prior to the following precipitation. This might result in more consistent yields from prep to prep and is highly recommended for carbonate containing samples.

  6. Add 150 µL Buffer SL3 and vortex for 5 s. Incubate for 5 min at 0–4 °C.

  7. Centrifuge for 1 min at 11,000 x g.

  8. Place the NucleoSpin® Inhibitor Removal Plate onto the MN Square-well Block. Load up to 1 mL clear supernatant from step 4 to each well of the plate.

  9. Centrifuge at 5,600–6,000 x g for 5 min. Repeat loading step to process more than 1 mL of lysate.

  10. Discard NucleoSpin® Inhibitor Removal Plate. Add 250 µL Buffer SB to the flow-through in each well of the MN Square-well Block. Mix by pipetting up and down.

  11. Place the NucleoSpin® Soil Binding Plate on top of a new square-well or round-well block (not provided). Load binding mixtures from step 6 onto the binding plate. Centrifuge at 5,600–6,000 x g for 5 min. Discard flow-through.

  12. 1st Wash: Load 500 µL Buffer SB. Centrifuge at 5,600–6,000 x g for 2 min. Discard flow-through.

  13. 2nd Wash: Load 550 µL Buffer SW1. Centrifuge at 5,600–6,000 x g for 2 min. Discard flow-through.

  14. 3rd Wash: Load 700 µL Buffer SW2. Centrifuge at 5,600–6,000 x g for 2 min. Discard flow-through.

  15. 4th Wash: Load 700 µL Buffer SW2. Centrifuge at 5,600–6,000 x g for 5 min. Discard flow-through.

  16. Centrifuge at 5,600–6,000 x g for 15 min or incubate the plate for 20 min at 37 °C. Note: Ethanol in Buffer SW2 inhibits enzymatic reactions and has to be removed completely before eluting the DNA. Incubation at 37 °C is more effective to remove traces of wash buffer and should be preferred if possible.

  17. Place the NucleoSpin® Soil Binding Plate on an opened Rack of Tube Strips. Load 100–200 μL Buffer SE directly into the center of the silica membrane of each well. Incubate for 1 min.

  18. Centrifuge at 5,600–6,000 x g for 2 min to elute final DNA product. Note: Preheating Buffer SE to 70 °C or/ and incubating the entire plate at elevated temperature for 5 min can increase final yield significantly. Furthermore, two consecutive elution steps (e.g., 2 x 100 μL) yield more DNA than one elution step with 200 μL.

  19. Check DNA yields on the Synergy HTX.

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