IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-19

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Streptavidin Beads: Agarose and Magnetic Retrial

Early Morning

  • Simultaneously ran protocol using:
    • (A) magnetic beads - using same protocol again, to retry proteinase K digest for elution
    • (B) agarose beads:
binding capacity: 15-20ug of biotin/mL of agarose bead solution
  biotin's molecular weight: 244.31g/mol
  thus, binding capacity = (15e-6 to 30e-6)/244.31 = 61 to 122 nmoles/mL of bead solution, or 61 pmoles/uL

1680 pmoles of binding sites on a 40-uL rxn's worth of boxes + 36 pmoles of binding sites on a rxn-including-biotinylated-oligos' free oligos = 1716pmoles of binding sites

1716pmoles binding sites/(61pmoles/uL of beads) = 27.8uL of beads needed, round to 28uL
  • Incubations
    • Agarose Beads:
      • 28uL bead solution
      • 40uL rxn
      • 32uL 1x folding buffer
      • TOTAL: 100uL
    • Magnetic Beads:
      • 200uL bead solution
      • 40uL rxn
      • 60uL 1x folding buffer
      • TOTAL: 300uL
  • Wash Protocols
    • Pellet:
      • Agarose Beads:
        • Spin down for 1min @ 14,000rpm
      • Magnetic Beads:
        • Use MagnaRack
    • Pipette out wash-supernatant into tube; repeat this each wash.
    • Add 100uL of 1x folding buffer (30mM MgCl2, just noticed)
    • Repeat 3 times
  • Elution Protocols
    • Reconstitute beads in 98uL 1x folding buffer
    • Add 2uL of proteinase K (indeterminate concentration; 5mg lyophilized Qiagen's discontinued proteinase K was rehydrated in 260uL of dH2O, as per instructions on label)
    • Incubate overnight @ 37[[:Category:{{{1}}}|{{{1}}}]]

Late Afternoon

  • Manipulation of Elute and Washes
    • Agarose Beads:
      • Beads were spun down for 2min @ 14,000rpm.
      • 100uL of elute was removed to a clean tube and Speedvaced for ~1hr total (Speedvac was stopped and sample was examined after 30min, 20min, and 7 min to ensure that there was no over-Speedvacing)
      • Washes were Speedvaced for ~2hrs.
    • Magnetic Beads:
      • Beads were pelleted with MagnaRack
      • 100uL of elute was removed to a clean tube and Speedvaced for ~1hr total (Speedvac was stopped and sample was examined after 30min, 20min, and 7 min to ensure that there was no over-Speedvacing)
      • Washes were Speedvaced for ~2hrs.
  • Gel
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