TITLE: Co-extraction of RNA and DNA from soil and sediment samples
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes extracting RNA and DNA from soil and sediment samples. 2 g of soil or up to 5 g of sediment can be processed in one extraction. The protocol is based on the RNeasy PowerSoil Total RNA Kit but does not rely on any components of the kit. It also replaces the RNeasy PowerSoil DNA Elution kit. The JetStar 2.0 columns are replaced with the HiPure Columns from Invitrogen's PureLink HiPure Plasmid Miniprep Kit. A lot of the buffers can be found in the following patent https://patents.google.com/patent/US7459548B2/en.


[BEFORE_START]
Make sure all buffers are prepared before starting.

[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH. 
Consider working with two layers of gloves when working with phenol and change gloves often.  

[STEPS]
SECTION: Cell lysis
1. 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. 
 
   

SECTION: Cell lysis
2. Add up to 2 g of soil or up to 5 g of the sediment sample.
  

SECTION: Cell lysis
3. Add 2.5 mL , 0.25 mL , 0.8 mL and  3.5 mL pH 8  . Two layers will appear in the centrifuge tube. 
  

SECTION: Cell lysis
4.  Control that the cap is closed properly. Vortex until the biphasic layer disappears. 

SECTION: Cell lysis
5. Place the samples on a Vortex adapter (e.g. Qiagen) and vortex at maximum speed for 15 min .
  

SECTION: Cell lysis
6. 2500 x g, 10 min 

SECTION: Cell lysis
7. Transfer 3.5 mL 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.
    
 

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


SECTION: Inhibitor removal
9. Incubate at 4 °C for 10 min .

SECTION: Inhibitor removal
10. 2500 x g, 10 min 

SECTION: Inhibitor removal
11. Without disturbing the pellet (if there is one), transfer the supernatant to a new 15 mL centrifuge tube.
SECTION: Nucleic acid precipitation
12. Add 5 mL isopropanol. Mix by vortexing and incubate at Room temperature for 30 min .

SECTION: Nucleic acid precipitation
13. 2500 x g, 30 min .

SECTION: Nucleic acid precipitation
14. 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 5 min .
  


SECTION: Anion exchange clean-up
15. Add 1 mL to the pellet and incubate the sample at 45 °C  for 10 min . Afterwards, dissolve the pellet by vortexing and/or pipetting.

SECTION: Anion exchange clean-up
16. Place a HiPure or JetStar 2.0 column in a 15 mL centrifuge tube or a suitable rack.

 

 

SECTION: Anion exchange clean-up
17. Equilibrate the column with 2 mL . Wait until all of the buffer volume has passed the column by gravity flow. 

SECTION: Anion exchange clean-up
18. 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. 
SECTION: Anion exchange clean-up
19. Wash the column with 1 mL . Wait until all of the buffer volume has passed the column by gravity flow. 

SECTION: Anion exchange clean-up
20. Transfer the column to a new 15 mL centrifuge tube or place a 2 mL collection tube in the rack to collect the RNA.
SECTION: Anion exchange clean-up
21. Add 1 mL to the column. Wait until all of the buffer volume has passed the column by gravity flow. 

SECTION: Anion exchange clean-up
22. 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
SECTION: Anion exchange clean-up
23. Add 1 mL to the column. Wait until all of the buffer volume has passed the column by gravity flow. 
SECTION: Nucleic acid precipitation
24. If a 15 mL centrifuge tube was used for elution, transfer the DNA/RNA sample to a 2 mL tube.
SECTION: Nucleic acid precipitation
25. Add 1 mL of isopropanol and incubate at -20 °C  for 15 min 

SECTION: Nucleic acid precipitation
26. 13.000 x g, 15 min 

SECTION: Nucleic acid precipitation
27. Decant the supernatant and invert the tubes on a paper towel for 10 min to dry the DNA/RNA pellet. The RNA pellet should be barely visible and white to transparent, while the DNA pellet is mostly brownish.  
  

SECTION: Nucleic acid precipitation
28. Resuspend the DNA/RNA in 100 µL . Check the integrity of the DNA/RNA on an agarose gel. afterward