Revision as of 17:26, 21 October 2009

University of Alberta - BioBytes

Anchor Binding Capacity

Prepare a 20 μM solution of the Anchor (same as ____ 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

@@ Line 34: / Line 34: @@
 </ul>
-<h2>Binding Capacity: vary the amount of Anchor</h2>
+<h2>Binding Capacity</h2>
 <ul>
-<li>Make a series of 50 μL serial dilutions of 2 μM Anchor solution (1/2, 1/5, 1/10, 1/15, 1/20, 1/25, 1/30, etc)
+<li>Prepare a 20 μM solution of the Anchor (same as ____ but using 30 μL water instead of 80 μL)
-<li>Prepare a tube for each dilution of Anchor made, each containing 40 μL 4 mg mL<sup>-1</sup> beads.
+<li>Set up a set of microcentrifuge tubes with 10, 20, 30, 40, 50, 60, 70, 80 μL of 4 mg mL<sup>-1</sup> beads dispensed into the respectively labelled tubes.
 <li>Wash the beads twice with 100 μL Wash/Binding buffer and once with 100 μL water, then aspirate water off the beads.
-<li>Add 20 μL of each dilution to it's respective tube with washed beads.
+<li>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).
 <li>Let bind at room temperature for 10 minutes.
 <li>Take readings at A<sub>260</sub> 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)