1 Purification Kit Introduction & Required Tools

Any crude probe contains traces of salts, dyes and degenerated probes that may interfere with downstream applications. The OliPure spin column kit for dual labeled 6-FAM / BHQ1 probes is optimized to remove the above mentioned contaminants by spin column based hydrophobic-interaction-chromatography.

1.1 Lab Equipment

• Pipet which covers 40µL
• Centrifuge (suitable for 0.2mL and 1.5mL tubes)
• Vortex mixer
• Two 1.5mL reaction tubes
• One 0.2mL reaction tube

2 Step by Step Instruction

2.1 Clean-up of dual-labeled 6-FAM / BHQ1 probe

1. Take your 0.2mL tube with crude probe and resuspend it in 40µL of solvent #1 provided (0.1M TEAA in H2O).
2. Vortex for 2 minute and briefly spin down in centrifuge.
   The dissolved probe appears pale to deep purple and / or green
3. Take a spin column, unscrew its top, remove the red rubber on the bottom and place it in a 1.5mL tube.
4. Pipett the full 40µL of the probe into the spin column.
5. 1st Spin: Place the 1.5 ml tube with the lid open and the spin column inserted into your centrifuge. Centrifuge at 500 - 10,000 × g for minimum 10 sec.
   The eluent appears clear. Spin column appears pale purple and / or green; the probe has bond to the column.
6. Discard the eluent and place tube plus spin column back into the centrifuge.
7. 2nd Spin: Add 120µL of solvent #2 provided (25% ACN in aq. 0.1M TEAA) to the column and centrifuge at 500 - 10,000 × g for minimum 10sec.
   The eluent appears pale to strong green. Free 6-FAM and degenerated probes have eluted.
8. Discard the eluent and place tube plus spin column the back into the centrifuge.
9. 3rd Spin: Add 120µL of solvent #3 provided (destilled H2O) to the column and centrifuge at 500 - 10,000 × g for minimum 10 sec.
   The eluent appears clear.
10. Discard the eluent and the 1.5mL tube. Place the spin column in a fresh 1.5mL tube to collect the purified probe and place it into the centrifuge.
11. 4th spin: Add 40µL of solvent #4 provided (50% ACN in H2O) to the column and centrifuge at 500 - 10,000 × g for minimum 10sec. Discard the spin column after checking its colour.
    The eluted probe appears purple. The spin column appears white.
12. Remove the solvent by drying the probe, either by heating it to 50°C until dry or by transferring the probe to a 0.2mL reaction tube and using the drying protocol of the Kilobaser Synthesizer.

3 Quantification

Via microvolume spectrophotometer: For 6-FAM probes with BHQ-1, use following formula:

Concentration in