IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-9-17

Streptavidin Depletion Assay: Redux for Brightness

In order to increase the visibility of previous bands, I'll try the experiments done 8.31 again, with twice as much starting material (ie. 20uL instead of 10uL of each).

NB - Replacements:

• The original 1M MgCl2 solution couldn't be found - a mixture of 1M MgCl2 from the other lab room's shelves was used instead.
• No 1x TBE could be found, thus 5x TBE was diluted 1:10 for the gel and gel buffer at 0.5xTBE.

Protocol

Test Solutions

• a) c5.0.A lidless (barrel) - 20uL
• b) c5.0.9b (biotinylated oligos - inside) - 5uL + 15uL H2O
• c) c5.0.Eb (outside biotinylated barrels) - 20uL
• d) c5.0.Fb (inside biotinylated barrels) - 20uL

The folded reactions used were all folded 8.31.

Treatment Conditions

• (1) Untreated
• (2) Incubated with Beads
  • in four clean tubes, use the MagnaRack to pellet 150uL of magnetic streptavidin beads (NEB); remove the supernatant (ie. the initial solution they were suspended in)
  • pipette the 20uL of each test solution in to the bottom, repeatedly triturating with the beads and trying to avoid as much as possible leaving "beads" of liquid on the sides of the tube
  • let sit for >5 min
  • add 1x folding buffer, 30mM MgCl2, up to 30uL (ie. 10uL)
• (3) Incubated with free-streptavidin
  • in four clean tubes, put 10uL of each test solution in tube
  • triturate, avoiding "beading" as much as possible on the sides, with 3uL of 1mg/mL stock tube of streptavidin (NEB)
  • add 1x, 30mM MgCl2 folding buffer to 30uL (ie. 7uL)
  • let sit for >5min
• (4) Incubated with free-streptavidin, then beads
  • in four clean tubes, use the MagnaRack to pellet 100uL of magnetic streptavidin beads (NEB); remove the supernatant (ie. the initial solution they were suspended in)
  • in four separate clean tubes, repeat the steps done for (3) Incubated with free-streptavidin.
  • pipette the 30uL of the free-streptavidin-incubated solutions into the bottoms of each of the tubes of pelleted beads, repeatedly triturating with the beads and trying to avoid as much as possible leaving "beads" of liquid on the sides of the tube
  • let sit for >5 min

After completion of all of these steps, the tubes containing beads (ie. (2) and (4)) were pelleted using the MagnaRack. The supernatants of these tubes were loaded into the gel, as were the entire 10uL of the original untreated folded reactions and the 30uL of the free-streptavidin incubated reactions.

Refolding

• Stupid mistake made after loading entirety of gel: accidentally bumped box and lost ~1/4 of material in wells. Thus, in case gel shows no improvement from 8.31 gel in terms of brightness, folded more boxes for repeat of today's experiment.

• 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 loading dye each reaction
• Ran at 75V for 1hr30m in 0.5x TBE, 10mM MgCl2

lane	component	amount	treatment conditions
1	1kb+ ladder	10uL	-
2	p7308 (~42nM)	9.4uL	-
3	c5.0.A (barrel)	20uL	untreated
4	c5.0.9(b)r (biotinylated oligos, \"outside\")	10uL	untreated
5	c5.0.E(b)r (outside biotinylated barrel)	20uL	untreated
6	c5.0.F(b)r (inside biotinylated barrel)	20uL	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

• Gel bands somewhat brighter, but streak up - might be worth it to repeat again. Why do they streak?