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

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