0102030405
Spin Pathogen DNA/RNA Kit-HYC427
Built on silica-column purification technology, this workflow eliminates toxic phenol-chloroform extraction and time-consuming ethanol precipitation steps. Purified RNA is directly compatible with RT-PCR, Northern blotting, viral detection and other downstream assays. Purified DNA can be directly applied to conventional PCR, real-time quantitative PCR, viral detection and related analyses.
Components
|
Components |
50 T |
|
Homogenizer tubes C |
50 |
|
Viral Micro Columns |
50 |
|
2ml Collection Tubes |
50 |
|
Proteinase K Solution |
1.2 mL |
|
Buffer MLBN |
30 mL |
|
Buffer VHB* |
13 mL |
|
Buffer RW2* |
20 mL |
|
Nuclease Free Water |
10 mL |
Storage
This product can be stored at room temperature (15–25℃) for 18 months.
Instructions
Preparations
- Absolute Ethanol
- Centrifuge tubes
- RNase-free pipette tips
- Prior to use, dilute Buffer RW2 with absolute ethanol and store at room temperature.
- Prior to use, dilute Buffer VHB with absolute ethanol and store at room temperature.
Protocol
- Add 20 μL Proteinase K and 400–500 μL test sample (whole blood, serum, plasma, culture medium, secretion, swab eluate, tissue homogenate, etc.) into a 2 mL Homogenizer Tube C, then tightly cap the tube.
- Tissue samples:Cut tissues into small fragments, transfer 10–50 mg tissue into Homogenizer Tube C, then add 20 μL Proteinase K and 500 μL Buffer TE, Buffer PBS or normal saline.
- Large-volume samples:Transfer 1.5–5.0 mL plasma, ascites or effusion into a 2–5 mL centrifuge tube. Centrifuge at 13,000 × g for 10 min to pellet microorganisms and cells. Discard supernatant, retain 500 μL residual liquid or cell pellet, and vortex to resuspend for subsequent use.
- Sputum samples:Take an appropriate volume of sputum, add 2–4 volumes of 0.1% DTT for liquefaction. Centrifuge at 13,000 × g for 10 min to pellet microorganisms and cells, discard supernatant, retain 500 μL residual liquid or pellet, and vortex to resuspend before use.
- Perform bead-beating lysis using a vortex mixer or bead homogenizer per laboratory equipment availability:
- Vortex mixer: Secure the tube on the mixer, vortex at maximum speed for 5–10 min.
- PowerLyzer bead homogenizer: Run at 2000 rpm for 30 sec, pause for 30 sec, then repeat at 2000 rpm for another 30 sec.
- FastPrep24 bead homogenizer: Run at 5 m/s for 30 sec, pause for 30 sec, then repeat at 5 m/s for another 30 sec.
- TissueLyser II bead homogenizer: Run at 25 Hz for 5 min, reposition the rack, then run again at 25 Hz for another 5 min.
- Incubate the sample at room temperature or 55 °C for 10 min to complete further digestion, then centrifuge at 12,000 × g for 1 min.This centrifugation step is not required for serum, plasma, swab eluates and similar specimens.
- Transfer 250 μL homogenate into a 1.5 mL centrifuge tube, add 500 μL Buffer MLB, and vortex for 5–10 sec to mix thoroughly.
- Fit the Viral Micro Column onto a 2 mL collection tube. Load the entire lysate onto the column, then centrifuge at 10,000 × g for 30–60 sec.
- Discard flow-through and reattach the column to the collection tube. Add 500 μL ethanol-diluted Buffer VHB to the column, then centrifuge at 10,000 × g for 30–60 sec.
- Discard flow-through and reattach the column to the collection tube. Add 500 μL ethanol-diluted Buffer RW2 to the column, then centrifuge at 10,000 × g for 30–60 sec.
- Discard flow-through and reattach the column to the collection tube. Add another 500 μL ethanol-diluted Buffer RW2 to the column, then centrifuge at 10,000 × g for 30–60 sec.
- Discard flow-through and reattach the column to the collection tube. Centrifuge the empty column at 13,000 × g for 3 min to dry residual liquid.
- Transfer the dried column into a new 1.5 mL centrifuge tube. Load 30–100 μL Nuclease-Free Water directly onto the center of the silica membrane, incubate for 2 min, then centrifuge at 13,000 × g for 1 min.
- Discard the column and store purified DNA/RNA at −80 °C.