Team:Alberta/Project/BeadBindingCapacity

From 2009.igem.org

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</ul>
</ul>
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<h2>Binding Capacity: vary the amount of Anchor</h2>
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<h2>Binding Capacity</h2>
<ul>
<ul>
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<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)
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<li>Prepare a 20 μM solution of the Anchor (same as <a href=https://2009.igem.org/Team:Alberta/Project/AnchTermAnneal>"Annealing Anchor/Terminator"</a> but using 30 μL water instead of 80 μL)
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<li>Prepare a tube for each dilution of Anchor made, each containing 40 μL 4 mg mL<sup>-1</sup> beads.
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<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>Wash the beads twice with 100 μL Wash/Binding buffer and once with 100 μL water, then aspirate water off the beads.
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<li>Add 20 μL of each dilution to it's respective tube with washed beads.
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<li>Prepare 4 μL of the 20 μM Anchor solution to separate tubes (corresponding to which bead-containing tube they will be added to) 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>Add half of this solution to the tubes containing the washed beads. Retain the other half.
 +
<li>Suspend the beads in the DNA solution.
<li>Let bind at room temperature for 10 minutes.
<li>Let bind at room temperature for 10 minutes.
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<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)
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<li>Pellet the beads with a magnet and aspirate the solution.
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<li>Convert DNA measurements to pmol and plot pmol DNA versus mg of beads. The slope gives the binding capacity
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<li>Take readings at A<sub>260</sub> with a UV-Vis Spectrophotometer of the DNA solutions prior to binding to bead (the half you retained) and after binding to bead (the aspirated solution). (ng of DNA can also be found by in-gel band intensity quantification methods)
</ul>  
</ul>  
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<h3>Calculation</h3>
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<ul>
 +
<li>Using the A<sub>260</sub> readings, calculate respective ng of DNA by multiplying volume of DNA applied to beads.
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<li>Calculate the difference of DNA for each set of readings per bead volume to give ng of DNA bound to bead.
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<li>Substitute the ng DNA bound into the following equation to get pmol of DNA: <br><math>(ng DNA<sub>bound</sub> x 10<sup>9</sup>) / (21519.1 g mol<sup>-1</sup> x 10<sup>12</sup>)</math></br>
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<li>Divide this number by the μL of beads used to get binding capacity: <br><math>μL Beads x 10<sup>3</sup> x 4 mg mL<sup>-1</sup></math></br>
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</ul>
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<h3>Disclaimer</h3>
 +
<p>By doing the binding capacity in this manner we found that binding capacity is highly dependant on DNA and bead concentration (see the graphs <a href=https://2009.igem.org/Team:Alberta/DNAanchor>"here"</a>).  Thus, we simply used the calculated binding capacity for 40 μL of beads since this is the volume we use for all builds.  A better binding capacity assay should be developed, but due to time constraints this method proved good enough since multiple runs of this experiment yielded numbers with small standard deviation: <br>209 ± 20 pmol mg<sup>-1</sup></br>

Latest revision as of 20:06, 21 October 2009

University of Alberta - BioBytes










































































































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.
  • Prepare 4 μL of the 20 μM Anchor solution to separate tubes (corresponding to which bead-containing tube they will be added to) 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).
  • Add half of this solution to the tubes containing the washed beads. Retain the other half.
  • Suspend the beads in the DNA solution.
  • Let bind at room temperature for 10 minutes.
  • Pellet the beads with a magnet and aspirate the solution.
  • Take readings at A260 with a UV-Vis Spectrophotometer of the DNA solutions prior to binding to bead (the half you retained) and after binding to bead (the aspirated solution). (ng of DNA can also be found by in-gel band intensity quantification methods)

Calculation

  • Using the A260 readings, calculate respective ng of DNA by multiplying volume of DNA applied to beads.
  • Calculate the difference of DNA for each set of readings per bead volume to give ng of DNA bound to bead.
  • Substitute the ng DNA bound into the following equation to get pmol of DNA:
    (ng DNAbound x 109) / (21519.1 g mol-1 x 1012)
  • Divide this number by the μL of beads used to get binding capacity:
    μL Beads x 103 x 4 mg mL-1

Disclaimer

By doing the binding capacity in this manner we found that binding capacity is highly dependant on DNA and bead concentration (see the graphs "here"). Thus, we simply used the calculated binding capacity for 40 μL of beads since this is the volume we use for all builds. A better binding capacity assay should be developed, but due to time constraints this method proved good enough since multiple runs of this experiment yielded numbers with small standard deviation:
209 ± 20 pmol mg-1

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