Personal Interactome project | Barcoded primer generation
Enriching ssDNA with biotin for primer usage.
Purifying ssDNA following Dynabeads M-270 Streptavidin with minor changes.
Protocol based on : BIOTECHNIQUESVOL. 61, NO. 3 BIOFEEDBACK [Letter to the Editor] NaOH concentration and streptavidin bead type are key factors for optimal DNA aptamer strand separation and isolation Geoffrey K. Kilili, Lianna Tilton & Christine M. Karbiwnyk
but with change to NaOH step due to enriching the other strand compared to them.
Protocol
1 Buffer preparation
List of NaOH paramaters tested.
| Reagent | Volume per 10 mL | Final conc. | Memo _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ |
|---|---|---|---|
| 1M Tris-HCl (pH = 7.5) | 100 µL | 10mM | |
| 1M EDTA | 10 µL | 1mM | |
| 2.5 M NaCl | 4 mL | 2 M | |
| 5 M NaOH | X | 0 - 150 mM | |
| ddH2O | 890 -X µL |
| NaOH final conc. | Volume of 5 M NaOH per 10 mL |
|---|---|
| 10 mM | 20 µL |
| 20 mM | 40 µL |
| 75 mM | 150 µL |
| 150 mM | 300 µL |
Wash DynabeadsTM magnetic beads
- Resuspend the beads in the vial (i.e. vortex for >30 sec, or tilt and rotate for 5 min).
- Transfer the desired volume of beads to a tube.
- Add an equal volume of Buffer, or at least 1 mL and resuspend.
- Place the tube on a magnet for 1 min and discard the supernatant.
- Remove the tube from the magnet and resuspend the washed beads in the same volume of Buffer as the initial volume of beads taken from the vial (step 2).
- Repeat steps 4–5 twice, for a total of 3 washes.
Immobilize nucleic acids
- Resuspend beads in 2X B&W Buffer to a final concentration of 5 μg/μL (twice original volume).
- To immobilize, add an equal volume of the biotinylated DNA/RNA in distilled water to dilute the NaCl concentration in the 2X B&W Buffer from 2 M to 1 M for optimal binding.
- Incubate for 15 min at room temperature using gentle rotation. Incubation time depends on the nucleic acid length: short oligonucleotides (<30 bases) require max. 10 min. DNA fragments up to 1 kb require 15 min.
- Separate the biotinylated DNA/RNA coated beads with a magnet for 2–3 min.
- Wash 2–3 times with a 1X B&W Buffer.
- Resuspend to the desired concentration. Binding is now complete. Resuspend the beads with the immobilized DNA/RNA fragment in a buffer with low salt concentration, suitable for downstream applications.