HiPure Microbiome DNA Kit — Host-Background Reduction Workflow Note
Cat. No. D314801 / D314802 / D314803
Protocol 2 focuses on host-background reduction and microbial DNA enrichment. Protocol 1 is included as a simplified total DNA extraction route for biological samples.
Transfer 1.0–1.8 ml liquid biological sample to a 2 ml tube, centrifuge at 13,000 × g for 10 min, carefully remove supernatant and leave about 0.7 ml liquid plus sediment for resuspension.
Special sample handling: sputum should be fully liquefied with 4–5 volumes of normal saline or PBS containing 0.1% DTT, then centrifuged at 10,000 × g for 10 min; remove supernatant, add 0.5 ml PBS and resuspend before continuing. Solid samples such as paste, tissue blocks or intestinal contents should be homogenized in 1 ml PBS, allowed to stand for 3 min, and 0.5 ml supernatant transferred forward. For whole blood, do not exceed 1.0 ml because larger inputs may produce excessive sediment. These sample-specific pretreatment steps may extend the standard timeline.
24 min
Cumulative 37 min
Host-cell lysis and microbial cell collection
Add 1.0 ml Buffer LBX1 and 5 μl Proteinase K, invert 10–15 s, incubate at room temperature for 10 min with occasional inversion, then centrifuge at 13,000 × g for 10 min. Remove supernatant and leave about 50 μl liquid plus sediment.
This is the host-background reduction stage: eukaryotic cells are lysed while microbial cells are retained for downstream processing.
17 min
Cumulative 54 min
DNase treatment of host DNA background
Add 200 μl DNase Buffer and 1 μl DNase I, mix by inversion and incubate at 37°C for 15 min with oscillation at 600–900 rpm.
This step is used to remove eukaryotic-cell DNA released during the enrichment module.
4 min
Cumulative 58 min
Microbial lysis setup and bead disruption
Add 200 μl Buffer TL and 15 μl Proteinase K, vortex thoroughly, transfer all liquid to the 2 ml Bead Tube and disrupt microbial cells by fast bead grinding for 60–90 s.
The standard timeline uses fast grinding. Vortex disruption at maximum speed for 10 min can extend this section.
4 min
Cumulative 62 min
Clarify microbial lysate
Centrifuge the bead tube at 13,000 × g for 3 min and prepare the clarified supernatant for binding adjustment.
Debris removal before column loading helps protect the silica membrane from particulate load.
2 min
Cumulative 64 min
MLB binding adjustment
Transfer 250 μl supernatant to a new centrifuge tube, add 500 μl Buffer MLB and invert 6–8 times to mix.
ACL-style direct lysis is not used here; this workflow uses MLB binding adjustment after microbial disruption.
2 min
Cumulative 66 min
Column binding
Transfer the adjusted mixture to a HiPure DNA Mini Column I and centrifuge at 10,000 × g for 1 min. Discard the flow-through.
This marks the start of the shared silica column purification path.
2 min
Cumulative 68 min
DCW1 wash
Add 500 μl Buffer DCW1 and centrifuge at 10,000 × g for 1 min. Discard the flow-through.
Confirm that ethanol has been added to DCW1 before use.
4 min
Cumulative 72 min
DCW2 wash ×2
Wash the column twice with 500 μl Buffer DCW2, centrifuging at 10,000 × g for 1 min each time and discarding flow-through between washes.
Confirm that ethanol has been added to DCW2 before use.
3 min
Cumulative 75 min
Dry spin
Centrifuge at 10,000 × g for 2 min to remove residual wash buffer.
This step reduces possible wash-buffer carryover into the eluate.
5 min
Cumulative 80 min
Elution
Place the column into a clean 1.5 ml tube. Add 30–50 μl Buffer AVE to the membrane, place at room temperature for 3 min and centrifuge at 10,000 × g for 1 min.
Elution volume follows Protocol 2 for enriched microbial DNA.
1 min
Cumulative 81 min
Store DNA
Discard the column and store DNA at 2–8°C for short-term use or at −20°C for long-term storage.
Typical processing time, standard fast-grinding route≈ 80–100 min
Route note
The displayed cumulative timeline uses the protocol-specified host-cell lysis, DNase treatment and column steps, with fast bead grinding used for microbial disruption. Vortex-based bead disruption, sputum liquefaction, solid-sample homogenization or difficult sample cleanup can extend the total time beyond the standard route.
Protocol 1 · direct total DNA route
2 min
Cumulative 2 min
Sample setup in bead tube
Add 150 μl Buffer TL and 20 μl Proteinase K to the 2 ml Bead Tube, then add 300 μl biological sample such as whole blood, plasma, body fluid, swab soaking solution or cell suspension.
Sputum requires upstream liquefaction and concentration; solid samples are introduced as a small paste, tissue block or intestinal-content input with Buffer AVE adjustment.
2 min
Cumulative 4 min
Microbial cell disruption
Vortex at maximum speed for 10 min, or use a bead grinding machine for fast grinding for 60–90 s. The standard timeline uses the fast grinding route.
For vortex-based disruption, add approximately 8–9 min to this section.
4 min
Cumulative 8 min
Clarify disrupted sample
Centrifuge the bead tube at 13,000 × g for 3 min and prepare to transfer the clarified supernatant.
This step separates debris from the DNA-containing lysate before binding adjustment.
2 min
Cumulative 10 min
MLB binding adjustment
Transfer 250 μl supernatant to a new centrifuge tube, add 500 μl Buffer MLB and invert 6–8 times to mix.
Binding conditions are established before the mixture is loaded onto the silica membrane.
2 min
Cumulative 12 min
Column binding
Transfer the full MLB-adjusted mixture to a HiPure DNA Mini Column I and centrifuge at 10,000 × g for 1 min. Discard the flow-through.
Complete loading of the adjusted mixture is included in the cumulative time.
2 min
Cumulative 14 min
DCW1 wash
Add 500 μl Buffer DCW1 to the column and centrifuge at 10,000 × g for 1 min. Discard the flow-through.
Confirm that ethanol has been added to Buffer DCW1 before use.
4 min
Cumulative 18 min
DCW2 wash ×2
Wash the column twice with 500 μl Buffer DCW2, centrifuging at 10,000 × g for 1 min each time and discarding flow-through between washes.
The repeated DCW2 wash is part of the standard purification path.
3 min
Cumulative 21 min
Dry spin
Centrifuge the column at 10,000 × g for 2 min to remove residual wash buffer.
Drying reduces the risk of ethanol carryover into the eluate.
5 min
Cumulative 26 min
Elution
Place the column in a clean 1.5 ml tube. Add 50 μl Buffer AVE to the membrane, place at room temperature for 3 min and centrifuge at 10,000 × g for 1 min.
Elution volume follows Protocol 1.
1 min
Cumulative 27 min
Store DNA
Discard the column and store DNA at 2–8°C for short-term use or at −20°C for long-term storage.
Typical processing time, standard fast-grinding route≈ 30–45 min
Route note
Protocol 1 is a direct total DNA extraction route from a 300 μl biological sample input after TL / Proteinase K treatment and bead disruption. It does not include the LBX1 host-cell lysis and DNase host-background reduction module used in Protocol 2.
How to Read This Note
1. Workflow structure
This workflow separates the direct total DNA extraction route from the large-volume microbial enrichment route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. Protocol 2 is presented first because it better represents the microbiome-specific host-background reduction logic of this kit, while Protocol 1 is retained as a simplified total DNA extraction path for biological samples.
2. Time interpretation
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifuge handling, bead-tube handling, supernatant / filtrate removal and column repositioning. For short protocol ranges, the timeline uses the midpoint. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path when the manual provides alternative handling formats, while the note and total-time range indicate where extended handling may apply. Cumulative time runs continuously from the first step to final elution across each selected protocol route.
3. Workflow characteristics
D3148 includes two different workflow logics. Protocol 1 is a direct total DNA extraction route based on bead disruption, MLB binding adjustment and silica column purification. Protocol 2 first concentrates microbial cells from a larger liquid input, lyses host/eukaryotic cells with Buffer LBX1, removes released host DNA with DNase treatment, then disrupts microbial cells before the shared silica column purification path. This makes Protocol 2 more suitable when microbial DNA enrichment from host-DNA-rich samples is the main objective.
4. Practical considerations
The key handling point in Protocol 2 is clean removal of host-cell lysate supernatant while retaining the microbial-cell sediment. The pellet should be thoroughly resuspended before DNase treatment and before bead-tube microbial disruption. For sputum, complete liquefaction upstream is usually more important than modifying downstream column wash steps. For whole blood, the manual limits the input to no more than 1.0 ml in the large-volume route because excessive sediment can interfere with handling.
