IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-23

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Verify folding of design 5 and 6

Ran gel to verify folding from earlier this week. Also comparing new scaffold with old scaffold.

design 5 and 6 folding, old and new scaffold comparison
Lane Contents
1 ladder
2 p7308 old
3 p7308 new
4 c5.0.A (old scaffold)
5 c5.0.A (new scaffold)
6 c5.0.C
7 c5.0.D
8 c6.0.A
9 c6.0.B
10 c6.0.C

PEG fractionation (from 8-20)

Also including 8% and 10% PEG fractionation for c6.0.A, c6.0.B, and c6.0.C


Streptavidin Depletion Assay

Plans

  • Possible Issues and Solutions
    • Too much DNA; even if a reasonable amount (say 70% of the biotinylated sites) are binding strep, there wouldn't be a noticeable difference between the bead-treated boxes and non-bead-treated boxes.
      • Lower amount of DNA used to half a reaction.
        • But the bands aren't that bright as it is, despite the iris being completely open, manual exposure, and use of 5uL of EtBr - possibly due to DNA leeching out during the long (>2hour) electrophoresis? Is that likely at all? But at the same time, my gels have been coming out fainter and fainter as I run longer each time, and yesterday it wasn't even left in buffer for any amount of time at all.
          • Run at higher voltage (100V), shorter time (1.5hr) - the difficulty in differentiating the folded from the scaffold might be due recently to the switchover to 0.5xTBE. Take a risk with the higher voltage - check on the gel box every 20 minutes to make sure no boiling is occuring, and scrape off the electrode wire each time.
    • Too little free streptavidin
      • Overload with free streptavidin - add from the original stock tube, which is likely safer (in more accurate-for-streptavidin-efficacy) buffer than the dilutions that have been made. This should push the equilibrium towards more complete binding of sites.
    • Biotinylated stuff not binding.
      • Check by running a control of biotinylated oligos (untreated, beaded, free streped, free then beaded) - there are large amounts of the original tube oligos, so run 40pmol of biotinylated sites (10uM biotinylated sites in the pre-working stock = 10pmoles/uL), or 5uL (to account for inefficient binding and pipetting error) in one lane and 40uL in another, just to make sure that the binding actually works.
        • If oligos don't show up in the untreated lane, try SYBR gold staining - ssDNA will show up better at low concentrations that way.
    • Suspicion that the magnetic beads may work better because there's no worry of accidentally loading part of the pellet and, thus, bead-bound material.
      • Use magnetic beads, concentrated with the MagnaRack initially, and run high concentrations (ie. low total volumes).

Folded:

  • 4 rxns each of:
    • c5.0.A lidless
    • c5.0.Eb
    • (4 rxns of c5.0.Fb remain from Katie's foldings on Monday)
    • Had to mix:
      • c5.0.Eb
      • c5.0.Fb


Strep-Deplet Assay Protocol

Test Solutions

 TEST SOLUTIONS
 --------------
 a) c5.0.A lidless (barrel) - 10uL
 b) c5.0.9b (biotinylated oligos - inside) - 10uL 
 c) c5.0.Eb (outside biotinylated barrels) - 5uL + 5uL H2O
 d) c5.0.Fb (inside biotinylated barrels) - 10uL (folded 8.22.06)

Protocol

 TEST CONDITIONS
 ---------------
 1. Untreated
 2. Beaded
     - pellet beads and remove initial solution they were packaged in
     - mix with each test solution
     - incubate 5 min
     - add 1x 30mM folding buffer to 40uL (ie. 30uL)
 3. Free-streptavidined
     - test solution in tube
     - mix with 3uL of 1mg/mL stock tube of streptavidin (from NEB)
     - incubate 5min
 4. Free-strep, then beads
     - all of (3)'s steps
     - mix with pelleted beads that had had their initial suspension solutions removed
     - incubate 5 min


Gel

  • Loaded 10uL of 1.Untreated and ~38uL of the other test conditions into wells of 2% agarose gel, 5uL EtBr, 0.5x TBE, 10mM MgCl2, 2uL of loading dye each. Ran @ 100V in 0.5x TBE, 10mM MgCl2 for 1hr.
Lane Component Test Condition Amount
1 1kb+ ladder - 10uL
2 p7308 (~42nM) - 9uL
3 barrel (lidless) Untreated 10uL
4 biotinylated oligos Untreated 10uL
5 outside biotinylated barrel Untreated 10uL
6 inside biotinylated barrel Untreated 10uL
7 barrel (lidless) Beaded ~38uL
8 biotinylated oligos Beaded ~38uL
9 outside biotinylated barrel Beaded ~38uL
10 inside biotinylated barrel Beaded ~38uL
11 barrel (lidless) Free-strep ~38uL
12 biotinylated oligos Free-strep ~38uL
13 outside biotinylated barrel Free-strep ~38uL
14 inside biotinylated barrel Free-strep ~38uL
15 barrel (lidless) Free, bead ~38uL
16 biotinylated oligos Free, bead ~38uL
17 outside biotinylated barrel Free, bead ~38uL
18 inside biotinylated barrel Free, bead ~38uL
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