IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-22

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Contents

Folding

Design 5

Realized that lid oligos were accidentally added in folding of c5.0.A on 8-20. Refolding c5.0.A to be just barrel oligos. Will use in PEG fractionation.


Working Stocks Description Pre-Working Stocks Water Total
c5.0Ano latches, no aptamers, no lids1 (86 μL), 4 (2.5 μL), 5 (2.5 μL), 6 (1.5 μL), 7 (1.5 μL)106 μL200 μL

Streptavidin Depletion Assay

Protocol

Testing Conditions:

  • (A) Unbeaded
  • (B) Beaded
  • (C) Free-streptavidin incubated
  • (D) Free-streptavidin incubated, then beaded

Test Solutions:

  • (1) c5.0.A (lidless barrel, no biotinylation)
  • (2) c5.0.Eb (lidless, outside biotinylation)
  • (3) c5.0.Fb (lidless, inside biotinylation)

Mix:

  • (B) Beaded
28uL agarose streptavidin beads
40uL of each test solution rxn

Spin down; remove supernatant to new tube.
Speedvac to ~40uL
Load 40uL in gel
  • (C) Free-streptavidin incubated
        • Free-streptavidin concentration needed
          • 1680pmoles on biotinylated boxes + 36pmoles of free biotinylated oligos = 1716pmoles = 1.7nmoles
          • free streptavidin @ 2uM = 2nmol/uL
          • thus, for safety, include 2uL of streptavidin
2uL of 2uM streptavidin
40uL of test solution
26uL of H2O

Speedvac to ~40uL
Load 40uL in gel
  • (D) Free-streptavidin incubated, then beaded
2uL of 2uM streptavidin
40uL of test solution

Wait 15 minutes

Add 28uL of agarose streptavidin beads
Wait 5 minutes

Spin down; remove supernatant to new tube.
Speedvac to ~40uL
Load 40uL in gel

Gel

  • Gel was 2% agarose in 0.5xTBE, 5uL EtBr, 10mM MgCl2. Ran for 1.5hr @ 80V in 0.5xTBE supplemented to 10mM MgCl2.
Lane Component Amount
11kb+ ladder10uL
2p7308 (~42nM)9uL
3untreated c5.0 barrel (lidless)~38uL
4untreated outside-biotinylated c5.0 barrel~38uL
5untreated inside-biotinylated c5.0 barrel~38uL
6agarose-beaded c5.0 barrel~38uL
7agarose-beaded outside-biotinylated c5.0 barrel~38uL
8agarose-beaded inside-biotinylated c5.0 barrel~38uL
9free-streptavidin-treated c5.0 barrel~38uL
10free-streptavidin-treated outside-biotinylated c5.0 barrel~38uL
11free-streptavidin-treated inside-biotinylated c5.0 barrel~38uL
12free-strep, then bead-treated c5.0 barrel~38uL
13free-strep, then bead-treated outside-biotinylated c5.0 barrel~38uL
14free-strep, then bead-treated inside-biotinylated c5.0 barrel~28uL
  • Preliminary Conclusions:
    • no difference seen after adding free-streptavidin, and then beading to the outside biotinylated box (lane 13)
      • possible not enough free-streptavidin used
    • also, no difference in gel band brightness from untreated to beaded - agarose beads either do not work as well as expected (maybe should use magnetic beads), or the amount used was too low
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