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

Early Morning
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
 * Simultaneously ran protocol using:
 * (A) magnetic beads - using same protocol again, to retry proteinase K digest for elution
 * (B) agarose beads:

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

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