Anchor Binding Capacity

What you will need:

• Paramagnetic Streptavidin Beads
• Annealed Anchor

Binding Capacity

• Prepare a 20 μM solution of the Anchor (same as Annealing Anchor/Terminator but using 30 μL water instead of 80 μL)
• Set up a set of microcentrifuge tubes with 10, 20, 30, 40, 50, 60, 70, 80 μL of 4 mg mL^-1 beads dispensed into the respectively labelled tubes.
• Wash the beads twice with 100 μL Wash/Binding buffer and once with 100 μL water, then aspirate water off the beads.
• Add 4 μL of the 20 μM Anchor solution to each tube along with enough water to bring the final solution volume up to half the dispensed bead volume. (i.e. to the 40 μL tube add 4 μL Anchor + 16 μL water).
• Let bind at room temperature for 10 minutes.
• Take readings at A₂₆₀ with a UV-Vis Spectrophotometer of the dilutions prior to binding to bead and after binding to bead. (ng of DNA can also be found by in-gel band intensity quantification methods)
• Convert DNA measurements to pmol and plot pmol DNA versus mg of beads. The slope gives the binding capacity