IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-25

Purification

PEG precipitation

• Used samples of 3.2.H and 3.2.I folded yesterday
• 20 μL of each, 190 mL water, 50 mL 20% PEG / 2.5 M NaCl in respective 500 μL PCR tubes

• gel analysis (2% agarose):
  • lanes 3 and 6 (reconstituted precipitate of H and I, respectively) appear to be free of oligos (compare to lanes 2 and 5, which are raw folding reaction), but yields are still very low, as evidenced by amount of nanostructures in supernatant (lanes 4 and 7)

Gel purification

• 20 mL each of 3.2.D and 3.2.E run on a 2% agarose gel
• nanostructure bands isolated and purified using Qiagen gel purification kit (good for fragments up to 10 kb according to manufacturer)
• will run on agarose gel tomorrow to determine purity

Silver staining experiments

• goal: to determine the minimum concentration of streptavidin that can be visualized with silver stain, and to note the times at which different concentrations become visible
• methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with Bio-Rad Silver Stain Plus Kit and protocol.
  • 35 min. in fixative solution
  • 2 x 10 min. wash in water
  • XX min. in staining solution
  • 15 min. in stopping solution

The streptavidin band appears at half the motility of the dye band (which was bleached to white after silver staining).

lane	streptavidin		water (μL)	10x Tris-gly loading dye (μL)
	(μL)	(fmols)
1	10 μL 4 μM	40,000	0	2
2	10 μL 2 μM	20,000	0	2
3	5 μL 2 μM	10,000	5	2
4	5 μL 1 μM	5,000	5	2
5	5 μL 0.5 μM	2,500	5	2
6	2 μL 0.5 μM	1,000	8	2
7	1 μL 0.5 μM	500	9	2
8	2.5 μL 0.1 μM	250	7.5	2
9	1 μL 0.1 μM	100	9	2
10	1 μL 0.05 μM	50	9	2

• results:
  • first three lanes (40k, 20k, 10k pmol) stain reliably
  • fourth and fifth lane lane (5k, 2.5k pmol) barely stains
  • the rest of the lanes (1.25k pmol and less) do not stain more than the background
  • this is disappointing (we can image 1 pmol of streptavidin with Coomassie blue)