- MagVigen™ FFPE DNA Capture Nanoparticles
- Tissue Lysis Buffer
- Proteinase K
- Proteinase K Buffer
- Wash Buffer 1 Stock
- Elution Buffer
- Magnetic Rack (NVIGEN Cat# A20006)
- Use your own protocol to remove paraffin from the FFPE samples.
- Check Tissue lysis buffer to make sure there is no precipitation. If there is, warm up to 40°C to dissolve it.
- Prepare wash buffer 1 solution:
Calculate needed quantity of wash buffer 1 according to table 1. Add 500 μl of Isopropanol to every 500 μl of Wash Buffer1 stock solution.
- Prepare Proteinase K solution:
Add proteinase K buffer to the tube containing proteinase K powder. Gently mix until proteinase K is dissolved. Store proteinase K solution in -20°C freezer.
- Add 200 μlor 400 μlTissue lysis buffer to the FFPE sample. Follow the reagent volumes in Table 1.
- Add prepared proteinase K solution to the tube, gently vortex to mix well and incubate for 30 min at 55°C, cool to room temperature.
- Incubate at 80°C for 15 minutes, then cool to room temperature.
- Add MagVigen™ DNA Capture nanoparticles to the lysis buffer. Vortex to mix well.
- Add Isopropanol to the lysis reaction, vortex to mix and
incubate at room temperature for 45 minutes.
- Put the reaction tube on a magnetic rack to pellet the beads until the solution is clear (about 10 minutes).
- Slowly remove the supernatant. Be careful not to take any beads, remove as much solution as possible.
- Take tube off magnet, add Wash Buffer1. Mix by pipetting or vortexing. Briefly spin down to bring solution to bottom of the tube. Pellet the beads on magnetic rack until solution is clear (about 3 minutes), then remove the solution.
- Add Wash Buffer 2 (80% Ethanol), mix by pipetting. Briefly spin down to bring solution to bottom of the tube. Pellet the beads on the magnetic rack, wait until solution is clear and remove the solution.
- Repeat step 11 for an additional wash with 80% Ethanol.
- Leave tubes on magnetic rack and air dry the pellet for ~1 minutes.
- Add desired amount of Elution buffer to the beads, pipette up and down until all pellet has redispersed completely.
- Set the tube on a magnetic stand for 1 minutes.
- Collect supernatant from the bottom of the microcentrifuge tube carefully without disturbing the pellet. The supernatant contains the extracted DNA.
Table 1. Reagent volume used in each step.
|Tissue Lysis Buffer||200||400|
|Proteinase K Solution||3||6|
|Wash Buffer 1||100||200|
|Wash Buffer 2 / 3 (80% Ethanol)||100||200|