Magnetic Pathogen DNA/RNA Kit-HYC422

Components

Storage

Store at room temperature (10–30 °C).

Required Reagents

Isopropanol, PBS Buffer.

Instructions

Preparations

1. Check Buffer LBS-A and Buffer ML for precipitates before use. If precipitates are observed, fully redissolve them by water bath incubation at 56 °C.
2. This kit is developed to isolate DNA and RNA from intact microbial cells. For optimal recovery of microbial nucleic acids, use fresh samples and avoid repeated freeze-thaw cycles. For short-term storage or transportation, keep samples at 2–8 °C; for long-term preservation, store frozen at −20 °C or −80 °C.
3. To prevent false results derived from contamination, maintain a clean workspace and wear protective clothing. Set up appropriate control samples for quality control. Take proper handling measures to minimize cross-contamination risks.Throughout extraction, use DNA/RNA-free pipette tips and consumables. Tighten all reagent caps immediately after use to avoid contamination. Before and after running automated nucleic acid extractors, wipe equipment with 75% ethanol and perform UV irradiation for decontamination.

Sample Preprocessing

1. Non-viscous liquid specimens (urine, pleural/peritoneal effusion, cerebrospinal fluid, etc.)
2. Transfer 400 μL raw sample directly for extraction.
3. Swab specimens (nasal, pharyngeal, anal swabs, etc.)
4. Wet swabs (preserved in storage solution): Vortex thoroughly, then transfer 400 μL suspension directly for extraction.
5. Dry swabs: Rotate the swab head in 0.5 mL PBS for at least 20 s. Squeeze the swab repeatedly against the tube wall before removal to elute maximum microbial suspension and minimize sample loss. Collect 400 μL liquid for subsequent extraction.
6. Sputum
7. Transfer approximately 500 μL sputum sample, add 2-fold volume (approx. 1 mL) of liquefaction reagent Buffer GB1, and incubate at 37 °C with shaking at 600 rpm for 15–30 min. For thick sputum, vortex thoroughly every 5 min until complete liquefaction. Transfer adequate liquefied sputum to a centrifuge tube, centrifuge at 12,000 rpm for 5 min and discard supernatant. Resuspend the pellet in 400 μL sputum supernatant.

Note: Sample volume may be adjusted proportionally with the volume of Buffer GB1. Buffer GB1 is not supplied in this kit. Incubate thin sputum for an optional 10–15 min; incubate thick sputum for a recommended 30 min, depending on liquefaction status.

4. Bronchoalveolar Lavage Fluid (BALF)
5. Clear BALF: No liquefaction required; take 400 μL sample directly for extraction.
6. BALF with trace viscous sputum: Centrifuge the total BALF sample to collect the viscous bottom layer (containing sputum, host cells and microbes). Process this pellet following the sputum liquefaction protocol above, then resuspend the pellet in 400 μL supernatant for extraction.
7. Blood
8. Serum or plasma: Take 400 μL directly for extraction.
9. Low-volume whole blood (less than 200 μL): Top up to 400 μL with PBS before extraction.
10. Large-volume whole blood: Treat with red blood cell lysis buffer prior to extraction.
11. Microbial culture samples
12. Maximum loading capacity: ≤1×10⁹ bacterial cells or ≤1×10⁹ fungal cells.

Operating Protocol

1. Add 400 μL sample, 200 μL Buffer LBS-A and 20 μL Proteinase K into Lysis Tubes II, then vortex thoroughly. Complete lysis via one of the following shaking methods:
2. Vortex Lysis Tubes II at ambient temperature for 10 min.
3. Incubate Lysis Tubes II on a thermomixer at 65 °C with shaking at 2,500 rpm for 10 min (heating is optional if temperature adjustment is unavailable).
4. Load Lysis Tubes II onto a tissue homogenizer and run a compatible lysis program per instrument brand. For MP FastPrep-24-5G: shake at 6 M/S for 30 sec, followed by a 30 sec rest, repeat for 6 cycles in total.
5. Add 250 μL Buffer ML and 20 μL Proteinase K to the lysate in Lysis Tubes II, vortex to mix evenly. Incubate in a 56 °C water bath for 10 min, or on a thermomixer at 56 °C with 0 rpm for 10 min. Centrifuge at 12,000 rpm for 1 min at ambient temperature, then transfer all supernatant to a new centrifuge tube.
6. Add 400 μL isopropanol to the tube and vortex thoroughly. Perform a brief spin-down for 1–3 sec to collect liquid residue on the tube cap.
7. Add 30 μL Magbeads PN (fully resuspend beads before pipetting), vortex to homogenize. Incubate the tube on a thermomixer with shaking at 1,500 rpm for 3 min, then let stand for 1 min. Spin down briefly for 1–3 sec, place the tube on a magnetic rack and wait 1 min. Carefully aspirate and discard all supernatant with a pipette.
8. Remove the tube from the magnetic rack, add 800 μL Buffer GW1, and vortex at ambient temperature for 1 min (or shake on a thermomixer at 2,500 rpm for 1 min, same operation for all subsequent washing steps). Let stand for 1 min, spin down briefly for 1–3 sec, then place on the magnetic rack for 1 min or until beads are fully captured. Carefully aspirate all supernatant.
9. Repeat Step 5 once.
10. Take the tube off the magnetic rack, add 800 μL Buffer GW2 and vortex at ambient temperature for 1 min. Let stand for 1 min, spin down briefly for 1–3 sec, place on the magnetic rack for 1 min or until full bead capture, then aspirate all supernatant completely.
11. Repeat Step 7 once.
12. Leave the tube uncapped to air-dry for approximately 3 min until beads turn matte with no visible gloss. Add 70 μL RNase-Free Water and vortex for 1 min to resuspend beads. Perform a brief spin-down for 1–3 sec, place the tube on the magnetic rack and rest for 3–5 min until all magnetic beads are fully adsorbed.
13. Transfer the eluted nucleic acid to a new 1.5 mL centrifuge tube. The eluate can be directly applied to downstream experiments or stored long-term at −20 °C.