F-2 FECES PROCESSING
REDI-NET Consortium
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 feces processing.
Before start
BEFORE START
- Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
- Pre-heat the heater mixer at
95°C. - 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 Thermo Scientific Screw Cap Micro 1.5 ml 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.
- MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for
0h 3m 0s(before first use) or0h 1m 0s(before subsequent uses) to ensure that the magnetic silica particles are fully resuspended. - Aliquot nuclease-free water in big bottle into a few 15 ml tubes for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.
Steps
1. SAMPLE LYSIS
Add 700µL of ASL buffer to the bead tubes prepared in step 4 from Before Start under the Guidelines & Warning tab.
Use a clean spatula or forceps to weigh about 100mg fecal sample (roughly 5 drops of mouse feces) and place it into each prepared bead tube (If fecal sample is frozen, thaw and keep it On ice). Record the weight.
Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard Material, 100µL EBV, and 100µL ZIKV standard into a tube from step 4 from Before Start. Add 250µL ASL buffer.
Include a negative control for each batch of samples: a bead tube from step 4 from Before Start with 500µL ASL buffer only.
Refill the dry ice compartment of the Bullet Blender if necessary. Load all samples into the Bullet Blender.
Set the speed at 12 and the time at 3. Press Start.
After beating, incubate samples at 95°C for 0h 15m 0s.
After incubation, beat the samples in the Bullet Blender (Refill the dry ice compartment if necessary) at speed 12 and time at 3.
Let the samples settle for 0h 1m 0s in the Bullet Blender and then repeat step 8.
2. INSTRUMENT SET UP
Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument.
3. SET UP THE PROCESSING PLATES
Set up the Tip Comb, Wash, and Elution Plates outside the instrument according to the following table.
| 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 | 985 µL |
4. EXTRACTION
Centrifuge the bead tubes with lysate from step 9 for 12000x g.
Add 20µL of Proteinase K into wells of a Deep-well plate (based on the number of samples).
Transfer 270µL supernatant without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate.
Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL 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 (it can be mixed on a Hula mixer for 0h 2m 0s). Add 695µL mixture to each sample. Each well including sample lysate should be 985µL.
Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
Start the run, then load the prepared plates into position when prompted by the instrument.
5. 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 1µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.
Proceed with sample testing following the REDI-NET SOP F-4 Fecal Testing or store at -20°C for less than 2 weeks.
6. INSTRUMENT SET UP
Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
7. 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 |
| F | Tip comb | ||
| G | Empty | ||
| H |
Set up the Elution Strip according to the table below:
| A | B | C | D |
|---|---|---|---|
| Strip ID | Row | Reagent | Volume per well |
| Elution | A | Nuclease-free water | 75 µL |
8. EXTRACTION
Centrifuge the bead tubes with lysate from step 9 for 12000x g.
Add 20µL of Proteinase K into wells of Row A (based on number of samples) of a new Deep-well plate.
Transfer 270µL supernatant without any particle carryover to the wells of the Deep-well Row A containing proteinase K. This row becomes the Sample Row.
Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL 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 695µL mixture to each sample. Each well including sample lysate should be 985µL.
Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
Start the run, then load the prepared plate and Elution Strip into position when prompted by the instrument.
9. 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 1µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.
Proceed with sample testing following the REDI-NET SOP F-4 Fecal Testing or store at -20°C for less than 2 weeks.
APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
DNA quantification:
According to the volume of sample used, add the 1x HS dsDNA Qubit Assay for a final volume of 200µL (i.e., if using 1µL of sample, add 199µL of 1x HS dsDNA Qubit Assay). Vortex for 5 - 10 seconds, then incubate for 0h 2m 0s at Room temperature before reading.
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 199µL of Qubit HS RNA Assay working solution and 1µL of the sample. Vortex for 5 - 10 seconds, then incubate for 0h 2m 0s at Room temperature before reading.
