| Directions for use |
Reagent Preparation:
- Working Binding Buffer: Add 40 ml isopropryl alcohol to 120 ml Binding Buffer to prepare the Working Binding Buffer solution.
- Working Clean Buffer: Add 30 ml isopropryl alcohol to 30 ml Clean Buffer to prepare the Working Clean Buffer solution.
- Working Wash Buffer: Add 96 ml isopropryl alcohol to 24 ml Wash Buffer to prepare the Working Wash Buffer solution.
Notes:
- Vortex magnetic beads before use.
- Use germ-free, nucleic-acid-free and nuclease-free centrifuge tubes and pipette tips.
- The component content of cell-free DNA is extremely low, we recommend using low nucleic acid adsorption centrifugal tubes for plasma storage, DNA extraction and DNA storage.
- Avoid repeated freeze/thaw cycles of reagents, samples and isolated DNA.
Reaction System:
Prepare the reaction system in a 15 ml or 50 ml centrifuge tube according to the table below.
| Component |
Plasma Volume |
| 0.5 ml |
1 ml |
2 ml |
4 ml |
10 ml |
| 20% SDS |
25 µl |
50 µl |
100 µl |
200 µl |
500 µl |
| Proteinase K |
15 µl |
30 µl |
60 µl |
120 µl |
300 µl |
| Working Binding Buffer |
0.75 ml |
1.5 ml |
3 ml |
6 ml |
15 ml |
| Magnetic Cell-Free Beads |
10 µl |
20 µl |
40 µl |
80 µl |
200 µl |
Assay Procedure:
- Set up a reaction system according to the table above.
- Vortex the tubes for 15 seconds, then leave at room temperature for 20 minutes. Invert the tube 3-5 times throughout this time period.
- Magnetic separation: Place the centrifuge tubes on a magnetic stand, then gently spin the tubes left and right by hand. Reverse the spin direction when the magnetic beads begin to aggregate towards the tube wall, and repeat 2-3 times. Ensure that any beads on the lid aggregate to the tube wall. Allow to stand for 2 minutes and ensure all beads are aggregated to the tube wall.
- Discard the supernatant from the opposite side of the magnetic beads, taking care not to remove the beads themselves. Take the tubes off the magnetic stand and add 1 ml of Working Clean Buffer (with isopropryl alcohol). Vortex for 15 seconds, then transfer to a new 1.5 ml centrifuge tube. If magnetic beads remain in the original tube, transfer the supernatant back to the original tube, wash, then transfer to the 1.5 ml centrifuge tube. Repeat Step 3 to carry out another magnetic separation.
- Discard the supernatant. Take the tubes off the magnetic stand and add 1 ml of Working Wash Buffer solution (with isopropyl alcohol). Vortex for 15 seconds, then repeat Step 3 to carry out another magnetic separation.
- Repeat Step 5.
- Discard the supernatant, including any liquid on the lid. It is recommended to use smaller size pipette tips to remove the supernatant thoroughly.
- Allow to stand and air-dry for 5-10 minutes.
- Add Elution Buffer according to the table below
| Component |
Original Plasma Volume |
| 0.5 ml |
1 ml |
2 ml |
4 ml |
10 ml |
| Elution Buffer |
20 µl |
30 µl |
50 µl |
75 µl |
200 µl |
Vortex the tubes for 5 minutes.
- Place the centrifuge tubes on the magnetic stand. Transfer the liquid into a new 1.5 ml low nucleic acid adsorption centrifuge tube, taking care not to remove the beads themselves.
- Store the isolated DNA at -20 °C.
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