100 preps
The sample is homogenized in the lysis buffer and the nucleic acid is released into the buffer. The lysis buffer contains a high concentration of guanidine. In this condition, the membrane absorbs nucleic acid by hydrogen bond and electrostatic physical and chemical action, while proteins and other impurities are not absorbed. The lysate is transferred to the adsorption column for filtration, and the nucleic acid filter membrane is washed to remove residual proteins and other impurities, and finally eluted by the low-salt buffer solution. The obtained nucleic acids can be directly used in downstream related experiments such as reverse transcription, PCR, RT-PCR, fluorescence quantitative PCR, second-generation sequencing and Northern hybridization.
Main components
The kit consists of the following components:
|
The name of the reagent |
Amount |
Component description |
|
Lysis Buffer |
50 ml |
Provide environment for lysing and binding to the column |
|
Wash Buffer |
24 ml |
Remove residual proteins and other impurities |
|
Elute Buffer |
6 ml |
Nuclease-free solution |
|
Spin Column |
50 pcs × 2 |
Adsorb viral nucleic acid and collect the filtrate |
Storage conditions
Store at room temperature (15-25°C) and transport at room temperature.
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 Lysis Buffer. 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 96 ml anhydrous ethanol to Wash Buffer, and store at room temperature.
Protocol
1. Add 500 μl of Lysis Buffer into a 1.5 ml RNase-free centrifuge tube (self-provided). If there are many samples, the Lysis Buffer can be pre-packed.
2. Add 200 μl of samples, mix thoroughly by vortexing (if the sample is less than 200 μl, fill it with normal saline to 200 μl).
3. Transfer the above mixture to the Spin Column (with Collection Tube). Centrifuged at 12,000×g for 1 min.
4. Discard the filtrate and put the Spin Column back into the 2 ml Collection Tube. Add 600 μl of Wash Buffer and centrifuge at 12,000×g for 30 sec, then discard the filtrate.
Note: make sure the correct amount of anhydrous ethanol has been added to the Wash Buffer.
5. Repeat step 4 once.
6. Centrifuge for 2 min at 12,000×g to dry the column membrane. Discard the filtrate and collection tube.
7. Transfer the Spin Column to a new 1.5 ml Collection Tube (provided by the kit), add 50 μl Eluent Buffer to the center of the membrane of the Spin Column, and place it at room temperature for 1 min. Centrifuge at 12,000×g for 1 min.
8. Discard the Spin Column, the obtained DNA/RNA can be directly used for subsequent detection, or be stored at -30 ~ -15°C for short-term storage or at -70°C for long-term storage.