IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-22: Difference between revisions

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===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.
* Gel was 2% agarose in 0.5xTBE, 5uL EtBr, 10mM MgCl2.  Ran for 1.5hr @ 80V in 0.5xTBE supplemented to 10mM MgCl2.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Amount'''
|-
| 1||1kb+ ladder||10uL
|-
| 2||p7308 (~42nM)||9uL
|-
| 3||untreated c5.0 barrel (lidless)||~38uL
|-
| 4||untreated outside-biotinylated c5.0 barrel||~38uL
|-
| 5||untreated inside-biotinylated c5.0 barrel||~38uL
|-
| 6||agarose-beaded c5.0 barrel||~38uL
|-
| 7||agarose-beaded outside-biotinylated c5.0 barrel||~38uL
|-
| 8||agarose-beaded inside-biotinylated c5.0 barrel||~38uL
|-
| 9||free-streptavidin-treated c5.0 barrel||~38uL
|-
| 10||free-streptavidin-treated outside-biotinylated c5.0 barrel||~38uL
|-
| 11||free-streptavidin-treated inside-biotinylated c5.0 barrel||~38uL
|-
| 12||free-strep, then bead-treated c5.0 barrel||~38uL
|-
| 13||free-strep, then bead-treated outside-biotinylated c5.0 barrel||~38uL
|-
| 14||free-strep, then bead-treated inside-biotinylated c5.0 barrel||~28uL
|}
<gallery>
Image:TC-20060822-strepdeplet-2h.jpg
</gallery>

Revision as of 08:53, 23 August 2006

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
c5.0A no latches, no aptamers, no lids 1 (86 μL), 4 (2.5 μL), 5 (2.5 μL), 6 (1.5 μL), 7 (1.5 μL) 106 μL 200 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
1 1kb+ ladder 10uL
2 p7308 (~42nM) 9uL
3 untreated c5.0 barrel (lidless) ~38uL
4 untreated outside-biotinylated c5.0 barrel ~38uL
5 untreated inside-biotinylated c5.0 barrel ~38uL
6 agarose-beaded c5.0 barrel ~38uL
7 agarose-beaded outside-biotinylated c5.0 barrel ~38uL
8 agarose-beaded inside-biotinylated c5.0 barrel ~38uL
9 free-streptavidin-treated c5.0 barrel ~38uL
10 free-streptavidin-treated outside-biotinylated c5.0 barrel ~38uL
11 free-streptavidin-treated inside-biotinylated c5.0 barrel ~38uL
12 free-strep, then bead-treated c5.0 barrel ~38uL
13 free-strep, then bead-treated outside-biotinylated c5.0 barrel ~38uL
14 free-strep, then bead-treated inside-biotinylated c5.0 barrel ~28uL
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