The kit adopts the magnetic particle purification technology based on superparamagnetism, which can minimize the risk of cross-contamination and improve the sensitivity and accuracy of detection. The operation time of the instrument is only 50 minutes.
Main components
The kit consists of the following components: (for a 200 prep kit)
|
The name of the reagent |
Amount |
Component description |
|
Buffer MVL |
45 ml |
Provide environment for lysing |
|
Buffer MB |
25 ml |
Provide environment for binding to the magnetic beads |
|
Buffer MW1 |
55 ml |
Remove residual proteins and other impurities |
|
MagV Beads |
5.2 ml |
Adsorb viral nucleic acid |
|
DEPC-Water |
15 ml |
DEPC - treated water, RNase - free |
|
Carrier RNA |
450 µl |
Capture trace nucleic acid |
|
Proteinase K |
1 ml × 2 |
Lyses proteins bound to nucleic acids |
Storage conditions
Store Carrier RNA and Proteinase K at -20°C, MagV Beads and DEPC-Water at 2-8°C, others at room temperature (RT, 15-25°C), and transport at RT.
Notes
1. Prepare your own RNase-free pipette tips, 1.5 ml RNase-free centrifuge tubes, centrifuge, etc.
2. The virus has a strong ability to infect, a variety of defense measures must be done before the operation.
3. Avoid repeated freezing-thawing of samples, otherwise the extracted viral RNA will be degraded and the extracted amount will decrease.
4. All operating procedures, if not specified, are carried out at room temperature (15-25°C).
5. When using this kit, please wear lab coat, disposable latex gloves, disposable masks and use RNase-free consumables to avoid RNase pollution to the greatest extent.
6. Please check whether there is crystal precipitation in the Buffer MVL. If there is crystal precipitation, place it at room temperature or 37°C until the crystal is dissolved. Mix it before use.
Before use:
Add 55 ml anhydrous ethanol to Buffer MW1, and store at RT.
Add 58 ml isopropanol to Buffer MB, and store at RT.
Prepare a 75% ethanol solution.
Protocol A: Manual Operation Process
1. Viral lysis. Different lysis procedures should be adopted for different experimental materials, as follows:
A. Lysis of virus in plasma, serum, cell-free body fluids, and viral stock solution: prepare 10-200 µl of plasma, serum, cell-free body fluids, or viral stock solution. If the initial amount is less than 200 µl, use PBS solution or DEPC-Water to make up to 200 µl.
B. Lysis of virus-infected tissues: prepare 10 mg of virus-infected tissues to be ground with liquid nitrogen, and add 200 µl of PBS solution or DEPC-Water to the ground tissues.
C. Throat swab (with preservation solution): Vortex vigorously for 1 min, take 200ul for experiment.
D. Throat swab (dry): Add 300 µl Buffer MVL and 10 µl Proteinase K, vortex vigorously for 15 sec, mix well. Incubate at 56°C for 10 min, centrifuge at the highest speed for 1 min, take 200 µl supernatant and add 2 µl Carrier RNA. Continue the experiment from step 4.
2. Add 200 µl Buffer MVL, 10 µl Proteinase K and 2 µl Carrier RNA, vortex vigorously for 15 sec, mix well.
3. Incubate at 56°C for 10 min.
4. Add 400 µl Buffer MB and 25 µl MagV Beads to the above mixture, mix well. Let stand for 3-5 minutes.
5. Transfer the above mixture to the magnetic separation rack, and let stand for 3-5 minutes to absorb the beads. Carefully discard all solutions.
6. Add 500 µl Buffer MW1. Vortex mix for 15 seconds. Transfer the reclosion to the new tube.
7. Transfer the above mixture to the magnetic separation rack, and let stand for 2 minutes to absorb the beads. Carefully discard all solutions.
8. Add 600 ul 75% ethanol and vortex the mixture for 15 sec.
9. Transfer the above mixture to the magnetic separation rack, and let stand for 2 minutes to absorb the beads. Carefully discard all solutions.
10. Repeat steps 7-9 once.
11. Centrifuge briefly to collect droplets from the wall of the tube. Transfer to the magnetic separation rack and carefully discard all solutions.
12. Air drying for 7-10 min.
13. Add 30~50 µl DEPC-Water, vortex to disperse magnetic beads. Let stand for 3-5 min, during which gently oscillate 1-2 times to accelerate nucleic acid dissolution.
14. Transfer to the magnetic separation rack and let stand for 3 min. Transfer the nucleic acid solution to a new 1.5 ml centrifuge tube.