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

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

• 

• 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