Universal Pathogen DNA/RNA Extraction Kit (250 Preps)
Cat. No.: BMG-VR6568-02
Size: 250 Preps
Description
The EZgeneTM Viral DNA/RNA mini kit provides an easy and reliable method for isolating total viral DNA/RNA from plasma, serum, nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates, and sputum. This procedure has been tested for isolating nucleic acids from COVID-19, Hepatitis A, Hepatitis C and HIV. The isolated RNA can be used for PCR, qRT-PCR and other downstream applications.
Storage and Stability
All components can be stored at room temperature (15-25°C). All kit components are guaranteed for 1 year from the date of purchase.
Kit Components
| Catalog# |
BMG-VR6568-01 |
BMG-VR6568-02 |
| Preps |
50 |
250 |
| Lytic Enzyme Mix |
600 µL |
3 mL |
| Buffer LYE |
25 mL |
90 mL |
| Proteinase K (25 mg/mL) |
1 mL |
5 mL |
| RNA Wash Buffer * |
12 mL |
50 mL |
| Buffer RB |
20 mL |
90 mL |
| L Solution (RNA Carrier) |
210 µL |
1,100 µL |
| DEPC-Treated ddH2O |
5 mL |
15 mL |
| Mini Column with collection tubes |
50 |
250 |
| Collection tubes |
4x50 |
4x250 |
| Vacuum connecters (Optional) |
50 |
250 |
Materials required but not supplied
- Tabletop microcentrifuge and 1.5 mL RNase free tubes.
- 100% ethanol and isopropanol.
Handling RNA
Since RNases are difficult to inactivate, only use RNase free consumables for experiment. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure.
- Aseptic technique should always be followed when extracting with RNA. Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination.
- Change gloves frequently and keep tubes closed whenever possible.
- Work through protocol carefully and quickly to avoid RNA degradation.
Viral RNA Isolation Protocol
The protocol is developed for processing 50-300 μL samples.
1. Pipet 10 µL Proteinase K, 4 µL L Solution, and 300 µL Buffer LYE to a 1.5 mL tube.
Calculate the number of samples to be processed and make master mix of proteinase K, L Solution and Buffer LYE.
2. Pipet 300 µL nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirates, or plasma, serum, into the 1.5 mL tube from Step 1. Mix well by vortexting for 10 seconds. Incubate at room temperature for 2-5 minutes to lyse the cells and viruses.
3. Add 600 µL isopropanol and mix well by vortexing for 5 seconds.
Note: Maintain the ratio of (Sample+ Buffer LYE): Isopropanol= 1:1
4. Transfer 600 µL of the sample from step 3 into a RNA column and centrifuge at 10,000 rpm for 30 seconds. Discard the collection tube contains the flow-through and transfer the column to a new collection tube. Apply the remaining sample to the column and spin at 10,000 rpm for 30 seconds.
5. Discard the collection tube with the flow-through and transfer the column to a new collection tube. Add 500 µL Buffer RB to the column and centrifuge at 10,000 rm for 30 seconds. Discard the collection tube contains the flow-through and transfer the column to a new collection tube.
6. Add 500 µL RNA Wash Buffer to the column and centrifuge at 10,000 rpm for 30 seconds. Discard the collection with the flow-through and transfer the column to a new collection tube.
7. Centrifuge the empty column at 14,000 rpm (maximum speed) for 2 min. It is critical to remove residual ethanol for optimal elution.
8. Transfer the RNA column to a RNase-free 1.5 mL tube, add 35-50 µL DEPC-treated water directly to the center of the column membrane and centrifuge at 10,000 rpm for 30 seconds. The viral RNA is in the flow-through liquid.
9. Optional: Add the eluent back to the column membrane for a second elution.
Note: The first elution normally yields 70% of the RNA while the second elution yields another 20-30% of the RNA bound to the column.
Note: The purified RNA should be put on ice for downstream application or store at -80°C or -20°C.
Vacuum manifold protocol
1. Prepare samples by following the protocol on page 3, step 1-3. Prepare vacuum manifold according to manufacturer’s instructions. Insert a disposable connecter to the manifold and a RNA column to the connecter.
2. Pipet 600 µL sample/LYE/Proteinase K/L Solution/Isopropanol mixture to the column. Switch on vacuum source to draw the sample through the column. Process the remaining sample mix as described.
3. Wash the column by adding 500 µL Buffer RB; draw the Buffer RB through the column by the vacuum force.
4. Wash the column by adding 500 µL RNA Wash Buffer; draw the wash buffer through the column by the vacuum force.
5. Turn off the vacuum according to manufacturer’s instruction. Transfer the column to the collection tube and proceed to step 7 on page 3.
