Co-extraction of RNA and DNA from soil and sediment samples

Prepare one 15 mL conical centrifuge tube per sample by adding 0.5 g of a) 0.1 mm glass beads, b) 0.5 mm glass beads, c) 1 mm zirconia/silica beads d) 2 mm zirconia beads.

Add up to 2 g of soil or up to 5 g of the sediment sample.

Add 2.5mL , 0.25mL , 0.8mL and 3.5mL 8 . Two layers will appear in the centrifuge tube.

Control that the cap is closed properly. Vortex until the biphasic layer disappears.

Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 0h 15m 0s .

2500x g

Transfer 3.5mL of the upper aqueous phase to a new 15 mL centrifuge tube. Make sure not to pierce the biphasic layer and to not transfer any liquid from the lower phenol phase. If this happens by accident, recentrifuge the sample and repeat. In soil high in organic matter the biphasic layer will be much thicker than in sediment samples.

Add 1.5mL . The mixture will be cloudy at first but will clear up after vortexing for a few seconds.

Incubate at 4°C for 0h 10m 0s .

2500x g

Without disturbing the pellet (if there is one), transfer the supernatant to a new 15 mL centrifuge tube.

Add 5mL isopropanol. Mix by vortexing and incubate at Room temperature for 0h 30m 0s .

2500x g .

After centrifugation, a brown pellet should form on the bottom of the tube. Decant the supernatant and invert the tube on a paper towel for 0h 5m 0s .

Add 1mL to the pellet and incubate the sample at 45°C for 0h 10m 0s . Afterwards, dissolve the pellet by vortexing and/or pipetting.

Place a HiPure or JetStar 2.0 column in a 15 mL centrifuge tube or a suitable rack.

Note

The columns seem to be identical. Unfortunately, they are not sold separately but the Kit with the HiPure mini columns is way more affordable than the original kit from Qiagen (approx. 120€ for 25 preps). We are working on a solution to clean and reuse these columns further reducing the costs of this protocol.

Equilibrate the column with 2mL . Wait until all of the buffer volume has passed the column by gravity flow.

Load the sample from step 15 on the equilibrated column. Make sure all that the pellet is dissolved properly or it might heavily reduce the flow-through and reduce the expected yield. Wait until all of the buffer volume has passed the column by gravity flow.

Wash the column with 1mL . Wait until all of the buffer volume has passed the column by gravity flow.

Transfer the column to a new 15 mL centrifuge tube or place a 2 mL collection tube in the rack to collect the RNA.

Add 1mL to the column. Wait until all of the buffer volume has passed the column by gravity flow.

Transfer the column to a new 15 mL centrifuge tube or place a new 2 mL collection tube in the rack to collect the DNA

Add 1mL to the column. Wait until all of the buffer volume has passed the column by gravity flow.

If a 15 mL centrifuge tube was used for elution, transfer the DNA/RNA sample to a 2 mL tube.

Add 1 mL of isopropanol and incubate at -20°C for 0h 15m 0s

13.000x g

Decant the supernatant and invert the tubes on a paper towel for 0h 10m 0s to dry the DNA/RNA pellet. The RNA pellet should be barely visible and white to transparent, while the DNA pellet is mostly brownish.

Resuspend the DNA/RNA in 100µL . Check the integrity of the DNA/RNA on an agarose gel. afterward

Citation

In the gel picture, 2 clear RNA bands and an HMW DNA band should be visible. For 5g of sediment sample, the protocol typically yields ~ 5µg of DNA and RNA.