TITLE: S-2 SOIL PROCESSING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details about soil processing.

[BEFORE_START]
BEFORE START
 
Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program. 
Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 1) to Clear RINO brand 1.5 ml screw-cap microcentrifuge tubes.*
For the first time use of IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% isopropanol to ACB as indicated on the bottles (Optional if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit).
Buffer ATL may form precipitates upon storage. If necessary, warm to 56 °C until the precipitates have fully dissolved. Prepare buffer ATL-DX: add 100 µL Reagent DX to 15 mL Buffer ATL. If smaller amounts are needed, transfer 1.5 mL of Buffer ATL into a sterile 2 ml vial and add 10 µL Reagent DX. Mix well, after addition of Reagent DX. After preparation, the mixture is stable for 6 months at Room temperature (15-25°C)** 
MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for 3 min (before first use) or 1 min (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.** 
 



[GUIDELINES]
OBJECTIVE

To outline procedures for total nucleic acid extraction from soil samples.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on procedures for total nucleic acid extraction from soil samples to provide materials for downstream library preparation and sequencing for pathogen detection. 

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
 

MAINTENANCE OF EQUIPMENT

Caution on RNA handling

RNases are very stable and difficult to inactivate and only minute amounts are sufficient to destroy RNA. 
Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure. 
Sample handling and extraction should be performed under an extraction hood and respecting Good Laboratory Practices. 
Use filter tips all the time.

Storage of the buffers from IndiMag pathogen kit

Proteinase K is stable for at least 1 year after delivery when stored at Room temperature (15-25°C). To store for more than 1 year or if ambient temperature often exceeds 25 °C, storage at 2-8 °C is recommended. Do not add Proteinase K directly to the Buffer VXL mixture! This can cause clogs or precipitates.
Precipitation may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 30 min at 37 °C, with occasional shaking.
Reconstituted Buffer AW1 can be stored at Room temperature (15-25°C) for up to 1 year. Mix well after adding Ethanol.
Buffer AVE is RNase-free upon delivery. It contains sodium azide, an antimicrobial agent that prevents growth of RNase-producing organisms. However, as this buffer does not contain any RNase degrading chemicals, it will not actively inhibit RNases introduced by inappropriate handling. When handling Buffer AVE, take extreme care to avoid contamination with RNases. Follow general precautions for working with RNA, such as frequent change of gloves and keeping tubes closed whenever possible.

QUALITY CONTROL

This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance.

APPENDICES


 APPENDIX 1. MEASURING SPOON FOR 0.1 MM BEATING BEADS

The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. One spoon equals to 100 uL.
 
APPENDIX 3. EXPECTED OUTCOMES 

 



 
 

[STEPS]
SECTION: 1. SAMPLE LYSIS
1. Add 320 µL of 1x PBS and 80 µL ATL-DX Buffer to the bead tubes prepared on step 3 of Before Start section under the Guidelines & Warnings tab.  
 

SECTION: 1. SAMPLE LYSIS
2. 
 
Use a clean spatula or forceps to weigh about 0.25 g soil sample and place it into each prepared bead tube. Record the weight.

SECTION: 1. SAMPLE LYSIS
3. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material and  µL EBV, and 100 µL HIV standard into a tube from step 3 of Before Start section. Add 162.5 µL 1xPBS.
SECTION: 1. SAMPLE LYSIS
4. Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 320 µL cold sterile 1xPBS only.

SECTION: 1. SAMPLE LYSIS
5. Add dry ice into the cooling compartment of Bullet Blender and then load the all bead tubes (samples and controls).
SECTION: 1. SAMPLE LYSIS
6. Set the speed at 12 and time at 3. Press Start.
SECTION: 1. SAMPLE LYSIS
7. Let the samples settle for 1 min and then repeat step 6. 
 


SECTION: 2. INSTRUMENT SET UP
8. 
 

Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
SECTION: 2. INSTRUMENT SET UP
9. Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument. 
 

SECTION: 3. SET UP THE PROCESSING PLATES
10. Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table. 
 
 
     Plate   ID      Plate   position      Plate   type      Reagent      Volume   per well        Tip comb      7      Place a 96 Deep-well Tip comb in a deep-well   plate        Elution      6      Deep-Well      Nuclease-free   water      75   µL        Wash 4      5      Deep-Well      100   % ethanol      750   µL        Wash 3      4      Deep-Well      80%   ethanol      750   µL        Wash 2      3      Deep-Well      Buffer   AW2      700   µL        Wash 1      2      Deep-Well      Buffer   AW1      700   µL        Sample       1      Sample   Lysate      Lysate   and lysis buffer      990   µL   

SECTION: 4. EXTRACTION
11. Centrifuge the bead tubes with lysate from step 7 for 12000 x g, 5 min. 

SECTION: 4. EXTRACTION
12. Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate. 
 

SECTION: 4. EXTRACTION
13. Transfer 270 µL supernatant without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate. 
 


SECTION: 4. EXTRACTION
14. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming (can be mixed on Hula mixer for 2 min). Add 695 µL mixture to each sample (Optional if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit).
SECTION: 4. EXTRACTION
15. Select the program IndiMag_Pathogen_KF_Flex_4wash or the program MagMAX_Microbiome_Liquid_Buccal_Flex on the instrument according to the kit used.
SECTION: 4. EXTRACTION
16. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: 5. BIND, WASH AND ELUTE (Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
17. Vortex magnetic beads vigorously and for each sample, transfer 20 µL beads to 500 µL Binding Buffer. Make the master mix for multiple samples with 10% overage. Mix the master mix by inverting, then place the master mix on a rocker until use (Do not vortex).

SECTION: 5. BIND, WASH AND ELUTE (Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
18. When prompted (approximately 20 minutes after the start of the protocol), remove the Sample plate from the instrument.
SECTION: 5. BIND, WASH AND ELUTE (Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
19. Invert Binding Bead mix prepared in step 17 to mix, then add 520 µL to each sample in the Sample Plate. Remix the Binding Bead mix frequently to ensure even distribution of beads to all samples.
SECTION: 5. BIND, WASH AND ELUTE (Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
20. Place the Sample Plate back onto the instrument, then start the run.
SECTION: 6. QUANTIFICATION AND STORAGE
21. After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
SECTION: 6. QUANTIFICATION AND STORAGE
22. In a 0.6 mL microcentrifuge tube, use 3 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions. 
 

SECTION: 6. QUANTIFICATION AND STORAGE
23. Proceed with sample testing following the REDI-NET SOP S-4 Soil Testing or store at -20 °C for less than 2 weeks.
 

SECTION: 7. INSTRUMENT SET UP
24. 
 

Confirm 12-tip magnetic heads and 12 well deep-well heat blocks are being used.
SECTION: 7. INSTRUMENT SET UP
25. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
 

SECTION: 8. SET UP THE SAMPLE PLATE AND ELUTION STRIP
26. Set up the Sample Plate according to the table below:  
     Row   ID      Plate   Row      Reagent      Volume   per well        Sample row      A      Lysate   and lysis buffer      985   µL        Wash 1      B      Buffer   AW1      700   µL        Wash 2      C      Buffer   AW2      700   µL        Wash 3      D      80   % ethanol      750   µL        Wash 4      E      100   % ethanol      750   µL        Tip Comb       F      Tip   comb                       G      Empty               H   

SECTION: 8. SET UP THE SAMPLE PLATE AND ELUTION STRIP
27. Set up the Elution Strip according to the table below: 
 
 
     Row   ID      Plate   Row      Reagent      Volume   per well        Elution         A      Nuclease-free   water      75   µL   

SECTION: 8. SET UP THE SAMPLE PLATE AND ELUTION STRIP
26.1. 
 
 
SECTION: 3. SET UP THE PROCESSING PLATES
10.1. 
 
 
SECTION: 8. SET UP THE SAMPLE PLATE AND ELUTION STRIP
27.1. 
 
 
SECTION: 9. EXTRACTION
28. Centrifuge the bead tubes with lysate from Sample Lysis step 7 for 12000 x g, 5 min. 

SECTION: 9. EXTRACTION
29. Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate. 
 

SECTION: 9. EXTRACTION
30. Transfer 270 µL supernatant without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate. 
 

SECTION: 9. EXTRACTION
31. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming. Add 695 µL mixture to each sample (Optional if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit).

SECTION: 9. EXTRACTION
32. Select the program IndiMag_Pathogen_KF_Duo_4wash or the program MagMAX_Microbiome_Soil_Duo on the instrument.
SECTION: 9. EXTRACTION
33. Start the run, then load the prepared plates into position when prompted by the instrument.

 

SECTION: 10. BIND, WASH AND ELUTE(Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
34. Vortex magnetic beads vigorously and for each sample, transfer 20 µL beads to 500 µL Binding Buffer. Make master mix for multiple samples with 10% overage. Mix the master mix by inverting, then place the master mix on a rocker until use (Do not vortex).
SECTION: 10. BIND, WASH AND ELUTE(Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
35. When prompted (approximately 20 min after the start of the protocol), remove the Sample plate from the instrument.

SECTION: 10. BIND, WASH AND ELUTE(Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
36. Invert Binding Bead mix prepared in step 35 to mix, then add 520 µL to each sample in the Sample Plate. Remix the Binding Bead mix frequently to ensure even distribution of beads to all samples.
SECTION: 10. BIND, WASH AND ELUTE(Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
37. Place the Sample Plate back onto the instrument, then start the run.
SECTION: 11. QUANTIFICATION AND STORAGE
38. After the running protocol is completed (~ 35 min), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.

SECTION: 11. QUANTIFICATION AND STORAGE
39. In a 0.6 mL microcentrifuge tube, use 3 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions. 
 

SECTION: 11. QUANTIFICATION AND STORAGE
40. Proceed with sample testing following the REDI-NET SOP S-4 Soil Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
41. DNA quantification: 
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 3 µL of sample, add 197 µL of 1x HS dsDNA Qubit Assay. Vortex for 5 - 10 seconds, then Incubate for 2 min at Room temperature before reading.

SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
42. RNA Quantification:
SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
42.1. In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
 
     Reagents      Volume/sample      Volume   for n+1 sample        Qubit RNA HS Assay buffer      199   µL      ….   µL        Qubit RNA HS Assay Dye      1   µL      ….   µL   
 
SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
42.2. In a new 0.6 ml tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Vortex for 5 - 10 seconds, then incubate for 2 min at Room temperature before reading.