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.
  • interactome/experiments/2019121211-2.txt
  • Last modified: 2019/12/11 14:36
  • by dan