Jan 09, 2024
S-2 SOIL PROCESSING
- REDI-NET Consortium1
- 1REDI-NET Consortium
Protocol Citation: REDI-NET Consortium 2024. S-2 SOIL PROCESSING. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4j12gmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 12, 2023
Last Modified: March 15, 2024
Protocol Integer ID: 81782
Keywords: soil processing, QUBIT FLUOROMETER, SAMPLE LYSIS, EXTRACTION
Funders Acknowledgement:
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: HT9425-23-C-0059
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093 and HT9425-23-C-0059. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
This protocol details about soil processing.
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
Note
NOTE: All study procedures must be conducted in compliance with national and local policies for prevention and control of COVID-19 infection.
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 00:30:00 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
Expected result
| A | B | C | D | E | F | |
| Sample | Amount | Sample condition | Elution volume | DNA conc. (ng/ul) | RNA conc. (ng/ul) | |
| Tick | 1 unfed adult or 10 nymphs | Frozen/live | 75 | 20 - 30 | 10 - 20 | |
| Leech | 50 ul/ 3x3 mm/ 1 swab | Blood meal/ tissue/ swab | 75 | 5 - 100 | 5 - 100 | |
| Soil | 0.25 - 0.3 g | Frozen/Fresh | 75 | <0.025 - 20 | <0.01 - 20 | |
| Water | 750 ml | Half of the membrane | 75 | <0.025 - 20 | <0.01 - 20 |
Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
| A | B | |
| Equipment | Mfg / Product # | |
| KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head or KingFisher™ Duo Prime Magnetic Particle Processor | ThermoFisher, 5400630 or ThermoFisher, 5400110 | |
| Bullet Blender 24 Gold | Next Advance, BB24-AU | |
| Qubit 4 Fluorometer | ThermoFisher, Q33238 | |
| Adjustable micropipettes | Locally sourced | |
| Multi-channel micropipettes | Locally sourced | |
| Vortex | Locally sourced | |
| Tube centrifuge | Locally sourced | |
| Plate centrifuge | Locally sourced | |
| Digital scale/balance | Locally sourced | |
| Thermo Heater Mixer | Locally sourced |
| A | B | C | |
| Material | Description | Mfg / Product # | |
| IndiMag Pathogen Kit (MagMAX Microbiome Ultra Nucleic Acid Isolation Kit can be used instead if already procured) | w/o plastics, 384 reactions | Indical Bioscience, SP947257 | |
| Buffer ATL (for IndiMag kit only) | 200 mL, Tissue Lysis Buffer | Qiagen, 19076 | |
| Reagent DX (for IndiMag kit only) | 1 mL, Antifoaming Reagent | Qiagen, 19088 | |
| Measuring Spoon 100 µL | RNase Free, pack of 10. reusable | Next Advance, MSP01-RNA | |
| KingFisher™ Deepwell 96 Plate | KingFisher | ThermoFisher, 95040450 | |
| KingFisher™ 96 KF microplate | KingFisher Flex ONLY | ThermoFisher, 97002540 | |
| KingFisher™ 96 tip comb for DW magnets | KingFisher Flex ONLY | ThermoFisher, 97002534 | |
| KingFisher™ Duo Prime 12-tip comb | KingFisher Duo Prime ONLY | ThermoFisher, 97003500 | |
| Elution Strip | KingFisher Duo Prime ONLY | ThermoFisher, 97003520 | |
| KingFisher™ Duo Cap for Elution Strip | KingFisher Duo Prime ONLY | ThermoFisher, 97003540 | |
| MicroAmp™ Clear Adhesive Film | KingFisher | ThermoFisher, 4306311 | |
| RNase-Free Microfuge Tubes | Nonstick, 1.5 mL | ThermoFisher, AM12450 | |
| RNase-Free Microfuge Tubes | Nonstick, 2.0 mL | ThermoFisher, AM12475 | |
| Clear RINO brand microcentrifuge tubes | 1.5 mL, screw-cap | Next Advance, TUBE1R5-S | |
| Zirconium oxide beads | 0.1 mm, 400 g | Fisher Scientific, 50-154-2950 | |
| Qubit™ 1X dsDNA HS Assay Kit | (consumable) | ThermoFisher, Q33230 | |
| Qubit™ RNA HS Assay Kit | (consumable) | ThermoFisher, Q32852 | |
| RNaseZap™ RNase Decontamination Solution | To remove RNase from working area | ThermoFisher, AM9780 | |
| ZymoBIOMICS Microbial Community Standard Material | For positive controls | Zymo Research, D6300 | |
| AcroMetrix HIV-1 Controls | For TNA extraction positive control | ThermoFisher, CLS430320-12EA | |
| Human gammaherpesvirus (EBV) positive control | For TNA extraction positive control | NMRC made | |
| Spatula | For use with samples | Locally sourced | |
| Ethanol | 100% (molecular biology grade) | Locally sourced | |
| Isopropanol (for IndiMag kit only) | 100% (molecular biology grade) | Locally sourced | |
| Nuclease-free Water | To elute total nucleic acids | Locally sourced | |
| Dry ice | To maintain cold chain during sample handling | Locally sourced | |
| Ice bucket | To contain the dry ice | Locally sourced | |
| Kimwipes | To dry material | Locally sourced | |
| Qubit Assay Tubes | For Qubit DNA/RNA measurement (consumable) | Thermo Fisher, Q32856 | |
| Falcon tubes | 15 mL and 50 mL | Locally sourced | |
| Data sheets | REDI-NET DCS SP-1 Sample Processing Form | REDI-NET Data Portal |
APPENDIX 4. SET-UP INSTRUCTIONS FOR BARCODE PRINTING
| A | B | C | |
| Equipment / Material | Description | Mfg / Product # | |
| Thermal Printer | Zebra ZD421T Desktop Dual Barcode Printer - 203 dpi | Uline, H-9581 | |
| Thermal Transfer Ribbon | For use with Zebra thermal printer; Desktop thermal transfer ribbons - wax/resin, 4.33” x 244 (12/case) | Uline, S-18466 | |
| Cryo-labels | 667 1.00" x 0.38" Cap & Wrap CryoLabel® w/0.375" Cap, Blanks, 1" Core
| Electronic Imaging Materials, #335774-COLOR | |
| Handheld scanner | To scan barcode | Zebra, LS2208-SR20001R-NA | |
| 123Scan Software | To scan barcodes | 123Scan software | |
| Laptop or desktop computer with Google Chrome and access to the REDI-NET data portal | To connect with the handheld scanner, the thermal printer and the REDI-NET Data Portal | Locally sourced |
Cryo-labels
Equipment
KingFisher™ Duo Prime Purification System
NAME
Purification System
TYPE
Thermo Scientific™
BRAND
5400110
SKU
LINK
Equipment
Bullet Blender 24 Gold (1.5 mL snap and screw cap tubes, 4°C cooling)
NAME
Blender
TYPE
Next Advance
BRAND
BB24-AU
SKU
LINK
Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
IndiMag Pathogen Kit w/o plastics (384 reactions)INDICAL BIOSCIENCECatalog #SP947257
Buffer ATL (tissue lysis buffer)QiagenCatalog #19076
Reagent DXQiagenCatalog #19088
Measuring Spoon 100 uL RNase Free pack of 10Next AdvanceCatalog #MSP01-RNA
KingFisher™ Plastics for 96 deep-well formatThermo Fisher ScientificCatalog #95040450
KingFisher MicroplateThermo Fisher ScientificCatalog #97002540
KingFisher™ Flex™ Systems Consumables, KingFisher 96 tip comb for DW magnetsThermo FisherCatalog #97002534
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, 12-tip comb, for Microtiter 96 Deepwell plateThermo FisherCatalog #97003500
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, Elution stripThermo FisherCatalog #97003520
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, KingFisher Duo Cap for elution stripThermo FisherCatalog #97003540
MicroAmp™ Clear Adhesive FilmThermo Fisher ScientificCatalog #4306311
Nonstick, RNase-free Microfuge Tubes, 1.5 mLThermo FisherCatalog #AM12450
Nonstick, RNase-free Microfuge Tubes, 2.0 mLThermo FisherCatalog #AM12475
Sterile Microcentrifuge Tube 1.5 mL (RINO®) 500/caseNext AdvanceCatalog #TUBE1R5-S
Bertin Corp 0.1mm Zirconium oxide beads (450g) (qty 500)Fisher ScientificCatalog #50-154-2950
Qubit 1X dsDNA High Sensitivity Assay KitThermo Fisher ScientificCatalog #Q33230
Qubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
RNaseZap®Thermo ScientificCatalog #AM9780
ZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
Qubit™ Assay TubesInvitrogen - Thermo FisherCatalog #Q32856
Safety warnings
RISK AND PERSONAL PROTECTION
- Caution should be taken while processing samples as some chemicals may be harmful. Please use a fume-hood when required to avoid inhaling harmful chemicals.
- Gloves should be worn all the time when handling samples.
- Decontaminants such as DNA/RNaZap could irritate the skin, avoid contact with skin while preparing the workbench for nucleic acid extractions.
Before start
BEFORE START
Note
NOTE:
To prevent contamination samples nucleic acid extraction and amplification (PCR) should be performed in separate rooms.
- 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 00:03:00 (before first use) or 00:01:00 (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.**
Note
*If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, transfer all the 0.1 mm beating beads into a new clear RINO tube brand 1.5 mL screw-cap microcentrifuge tube.
**Optional if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit.
1. SAMPLE LYSIS
1. SAMPLE LYSIS
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.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, add 800 µL of Lysis Buffer to the clear RINO 1.5mL prepared on step 3 of Before Start section.
Note
If soil is frozen, thaw and keep it On ice .
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.
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.
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.
Add dry ice into the cooling compartment of Bullet Blender and then load the all bead tubes (samples and controls).
Set the speed at 12 and time at 3. Press Start.
Let the samples settle for 00:01:00 and then repeat step 6.
Note
STOPPING POINT: lysed samples can be stored at 4 °C Overnight .
1m
2. INSTRUMENT SET UP
2. INSTRUMENT SET UP
Note
NOTE: KingFisher Flex only, if using KingFisher Duo Prime, Section 7
Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit ensure the program MagMax_Microbiome_Soil_Flex has been downloaded and loaded onto the instrument.
3. SET UP THE PROCESSING PLATES
3. SET UP THE PROCESSING PLATES
Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table.
Note
NOTE: DO NOT use the elution buffer provided by the kit for TNA elution. The ingredients in the elution buffer inhibit the downstream DNA sequencing efficiency.
| A | B | C | D | E | |
| 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 | |
Note
NOTE: If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, set up the Wash, Elution, Tip Comb Plates outside the instrument according to the following table.
4. EXTRACTION
4. EXTRACTION
Centrifuge the bead tubes with lysate from step 7 for 12000 x g, 00:05:00 .
5m
Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, add 40 µL of Proteinase K to each sample.
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.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, transfer as much supernatant as possible (up to 500 µL ).
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).
Select the program IndiMag_Pathogen_KF_Flex_4wash or the program MagMAX_Microbiome_Liquid_Buccal_Flex on the instrument according to the kit used.
Start the run, then load the prepared plates into position when prompted by the instrument.
5. BIND, WASH AND ELUTE (Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
5. BIND, WASH AND ELUTE (Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
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).
When prompted (approximately 20 minutes after the start of the protocol), remove the Sample plate from the instrument.
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.
Place the Sample Plate back onto the instrument, then start the run.
6. QUANTIFICATION AND STORAGE
6. QUANTIFICATION AND STORAGE
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.
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.
Note
Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit. (see Appendix 2 and Appendix 3)
Proceed with sample testing following the REDI-NET SOP S-4 Soil Testing or store at -20 °C for less than 2 weeks.
Note
For long-term storage the sample needs to be stored at -80 °C following the REDI-NET SOP S-3 Soil Storage.
7. INSTRUMENT SET UP
7. INSTRUMENT SET UP
Note
NOTE: KingFisher Duo Prime only, if using KingFisher Flex, go to section 2
Confirm 12-tip magnetic heads and 12 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit ensure the program MagMAX_Microbiome_Soil_Duo has been downloaded and loaded onto the instrument.
8. SET UP THE SAMPLE PLATE AND ELUTION STRIP
8. SET UP THE SAMPLE PLATE AND ELUTION STRIP
Set up the Sample Plate according to the table below:
| A | B | C | D | |
| 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 | ||||
Note
NOTE: If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, set up the Wash, Elution, Tip Comb Plates outside the instrument according to the following table.
Set up the Elution Strip according to the table below:
Note
NOTE: DO NOT use the elution buffer provided by the kit for TNA elution. The ingredients in the elution buffer inhibit the downstream DNA sequencing efficiency.
| A | B | C | D | |
| Row ID | Plate Row | Reagent | Volume per well | |
| Elution | A | Nuclease-free water | 75 µL |
Note
NOTE: If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, set up the Elution plates outside the instrument according to the table below:
9. EXTRACTION
9. EXTRACTION
5m
Centrifuge the bead tubes with lysate from Sample Lysis step 7 for 12000 x g, 00:05:00 .
5m
Add 20 µL of Proteinase K into wells (based on number of samples) of a new Deep-well plate.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, add 40 µL of Proteinase K to each sample.
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.
Note
If using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit, transfer as much supernatant as possible (up to 500 µL ).
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).
Select the program IndiMag_Pathogen_KF_Duo_4wash or the program MagMAX_Microbiome_Soil_Duo on the instrument.
Start the run, then load the prepared plates into position when prompted by the instrument.
Note
Keep the door open while extraction is in process. The chamber of the KingFisher Duo Prime is small. Closing the door makes the ethanol vapor restrained inside the chamber and increases the ethanol contamination.
10. BIND, WASH AND ELUTE(Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
10. BIND, WASH AND ELUTE(Only if using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit)
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).
When prompted (approximately 00:20:00 after the start of the protocol), remove the Sample plate from the instrument.
20m
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.
Place the Sample Plate back onto the instrument, then start the run.
11. QUANTIFICATION AND STORAGE
11. QUANTIFICATION AND STORAGE
After the running protocol is completed (~ 00:35:00 ), 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.
35m
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.
Note
Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit. (see Appendix 2 and Appendix 3)
Proceed with sample testing following the REDI-NET SOP S-4 Soil Testing or store at -20 °C for less than 2 weeks.
Note
For long-term storage the sample needs to be stored at -80°C following the REDI-NET SOP S-3 Soil Storage.
APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
2m
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 00:02:00 at Room temperature before reading.
2m
RNA Quantification:
In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
| A | B | C | |
| Reagents | Volume/sample | Volume for n+1 sample | |
| Qubit RNA HS Assay buffer | 199 µL | …. µL | |
| Qubit RNA HS Assay Dye | 1 µL | …. µL |
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 00:02:00 at Room temperature before reading.
2m
Protocol references
REFERENCES
1. Roey Angel, Eva Petrova, Ana Lara-Rodriguez (07/30/2020). Total Nucleic Acids Extraction from Soil. https://dx.doi.org/10.17504/protocols.io.bi46kgze
2. User Guide: Indical IndiMag Pathogen Kit user’s manual.
3. User Guide: MagMAX Microbiome Ultra Nucleic Acid Isolation kit, Applied Biosystems, Pub. No. MAN0018070 Rev. C.0.