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

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)

• NB: Noticed that there were errors in the pre-working stock table. See c5.0 Table of Working Stocks.

• • 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

• 

  Brighter Bands

• 

  Darker background (doesn't highlight the strange bright front midway up as much)