Team:Alberta/Project/BeadBindingCapacity

From 2009.igem.org

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     <h1>Linearizing BioByte Constructs by I-SceI Digestion</h1>
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     <h1>Anchor Binding Capacity</h1>
<h2>What you will need:</h2>
<h2>What you will need:</h2>
<ul>
<ul>
-
<li>I-SceI
+
<li>Paramagnetic Streptavidin Beads
-
     <li>Circular Biobyte Construct
+
     <li>Annealed Anchor
</ul>
</ul>
-
<h2>Digestion:</h2>
+
<h2>Binding Capacity: vary the amount of Anchor</h2>
-
For a 20 μL reaction:
 
<ul>
<ul>
-
<li>5 μL DNA sample
+
<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>2 μL NEB 10x I-SceI Buffer
+
<li>Prepare a tube for each dilution of Anchor made, each containing 40 μL 4 mg mL<sup>-1</sup> beads.
-
<li>2 μL 10x BSA
+
<li>Wash the beads twice with 100 μL Wash/Binding buffer and once with 100 μL water, then aspirate water off the beads.
-
<li>10.5 μL ddH2O
+
<li>Add 20 μL of each dilution to it's respective tube with washed beads.
-
<li>0.5 μL I-SceI (5U/μL)
+
<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)
 +
<li>Convert DNA measurements to pmol and plot pmol DNA versus mg of beads. The slope gives the binding capacity
</ul>  
</ul>  
-
<BR>Incubate at 37ºC for 1 hour
+
 

Revision as of 17:12, 21 October 2009

University of Alberta - BioBytes










































































































Anchor Binding Capacity

What you will need:

  • Paramagnetic Streptavidin Beads
  • Annealed Anchor

Binding Capacity: vary the amount of Anchor

  • 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)
  • Prepare a tube for each dilution of Anchor made, each containing 40 μL 4 mg mL-1 beads.
  • Wash the beads twice with 100 μL Wash/Binding buffer and once with 100 μL water, then aspirate water off the beads.
  • Add 20 μL of each dilution to it's respective tube with washed beads.
  • 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

Retrieved from "http://2009.igem.org/Team:Alberta/Project/BeadBindingCapacity"