IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-19: Difference between revisions
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Latest revision as of 16:56, 19 August 2006
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
- Agarose Beads:
- Magnetic Beads:
- 200uL bead solution
- 40uL rxn
- 60uL 1x folding buffer
- TOTAL: 300uL
- Magnetic Beads:
- Wash Protocols
- Pellet:
- Agarose Beads:
- Spin down for 1min @ 14,000rpm
- Magnetic Beads:
- Use MagnaRack
- Agarose Beads:
- Pipette out wash-supernatant into tube; repeat this each wash.
- Add 100uL of 1x folding buffer (30mM MgCl2, just noticed)
- Repeat 3 times
- Pellet:
- 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.
- Agarose Beads:
- Gel