IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-31

Streptavidin Depletion Assay

Biotinylated oligos arrived today - yay! Proceeded according to previously written procedure.

Reconstitution

oligo name	nmoles in tube	EB necessary for 200uM (uL)
c5.0.8.1ra	79.7	398.5
c5.0.8.2ra	144.4	722
c5.0.8.5ra	136.9	684.5
c5.0.9.1ra	138.4	692
c5.0.9.2ra	55.8	279
c5.0.9.3ra	124.7	623.5
cc5.0.9.4ra	121.3	606.5
c5.0.9.5ra	162.7	813.5

Stocks Made

• Pre-Working Stock

• • Added these oligos in the following proportions to respective tubes for c5.0.8(b)r and c5.0.9(b)r
    • 2.5uL of each (biotinylated and unbiotinylated) c5.0.8.#r and c5.0.9.#r oligo in respective tubes
    • 7.5uL of dH2O for each (8*7.5 for c5.0.8 = 60uL, 8*7.5 for c5.0.9 = 60uL)

• Working Stocks c5.0.E(b)r"' (outside biotinylation) and c5.0.F(b)r"' (inside biotinylation)

• • Made as given here.

Folding

• Folded, using FOLDINGD protocol, 3 rxns each of A (lidless), E(b), F(b).

Per Reaction:
-----------
9.4uL of p7308 ~42nM
11uL of H2O
4uL of 10x folding buffer, 30mM MgCl2
16uL of working stock

Gel

• 2% agarose, 0.5x TBE, 10mM MgCl2, 5uL EtBr
• 2ul of 10x TBE/glycerol dye each reaction
• Ran at 75V for 1hr30m in 0.5x TBE

lane	component	amount	treatment conditions
1	1kb+ ladder	10uL	-
2	p7308 (~42nM)	9.4uL	-
3	c5.0.A (barrel)	10uL	untreated
4	c5.0.9(b)r (biotinylated oligos, \"outside\")	10uL	untreated
5	c5.0.E(b)r (outside biotinylated barrel)	10uL	untreated
6	c5.0.F(b)r (inside biotinylated barrel)	10uL	untreated
7	barrel	~30uL	incubated with streptavidin beads
8	biotinylated oligos	~30uL	incubated with streptavidin beads
9	outside-biotinylated barrel	~30uL	incubated with streptavidin beads
10	inside-biotinylated barrel	~30uL	incubated with streptavidin beads
11	barrel	~30uL	incubated with free streptavidin
12	biotinylated oligos	~30uL	incubated with free streptavidin
13	outside-biotinylated barrel	~30uL	incubated with free streptavidin
14	inside-biotinylated barrel	~30uL	incubated with free streptavidin
15	barrel	~30uL	incubated with free streptavidin, then with streptavidin beads
16	biotinylated oligos	~30uL	incubated with free streptavidin, then with streptavidin beads
17	outside-biotinylated barrel	~30uL	incubated with free streptavidin, then with streptavidin beads
18	inside-biotinylated barrel	~30uL	incubated with free streptavidin, then with streptavidin beads

• 

Key Points of Gel

• Lane 9 (bead-treated outside-biotinylated barrel) shows no band, whereas Lane 10 (bead-treated inside-biotinylated barrel) does! This means that the inside-biotins were protected from the streptavidins bound to the beads, but the outside-biotins were not.
• Lane 17 (free-streptavidin, then bead-treated outside-biotinylated barrel) show a band, meaning that the free streptavidin did bind to the outside-biotins. They thus blocked them from being bound by the bead-streptavidins, which would have kept them out of the supernatant and thus caused no band to be present in Lane 17.

SUCCESS! FINALLY! The protection assay worked and showed that inside biotins were protected by the barrel!"'