IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-5

Superconcentration Experiments

PAGE Coomassie Gel Imaging of Superconcentration of Eb, Fb, and Gb

• destained: 10 min in H2O, two times
• imaged gel from yesterday

lane	component	concentration	amount of component in well μL	streptavidin added μL
1	streptavidin	2uM	3.5	-
2	p7308	44uM	7.5	3.5
3	c4.0.6b (ie. biotinylated inside-oligos)	50nM each oligo (as in all pre-working stocks)	7	3.5
4	Eb PEG-pellet	21/8 of a rxn	15	3.5
5	Fb PEG-pellet	21/8 of a rxn	15	3.5
6	Gb PEG-pellet	21/8 of a rxn	15	3.5
7	streptavidin	2uM	7	-

12% PAGE run at 100V for 1hr10m coomassie-stained overnight

• Conclusions:
  • streptavidin band clear
  • biotinylated oligos coomassie band clear too, in different place
    • there are definitely streptavidin-bound biotinylated oligos in lane 3
  • it is difficult to see in the image, but a darker blue band was visible at the top of lane 5, where the nanostructure should be
    • streptavidin bound to Eb
    • unfortunately, Eb = inside-biotinylation -latches, so how the streptavidin bound and was visualized when the Gb (outside-biotinylation +latch1) didn't bind it is questionable
    • it is a very faint band
  • no bands were seen at the top of the gel in the expected lane 6 (Gb)

• is it possible that streptavidin actually bound to nanostructures can't be stained w/ coomassie?
  • that would explain why the inside-biotinylated +latch1 design, which would not (we expect) permit the biotinylated-oligos within to bind streptavidin, shows a lot more unbound streptavidin and biotinylated-oligos-bound streptavidin

PAGE EtBr Gel Imaging of Superconcentration of Eb, Fb, and Gb

• as a first try, the gel was destained a further 30min in H2O before EtBr staining was attempted
• 200mL of H2O +7uL of EtBr were used as a stain; the gel was allowed to stain for 15 min

• no luck, no bands at all were seen under UV
  • will save gel in H2O till figure out if there is anything to be done about it

More Folding Reactions for Further Superconcentration Experiments with Stains-All

• made working stocks (=12.5 rxns) for:
  • outside-biotinylation w/o latches, Eb
  • outside-biotinylation w/ "future" (aka four-oligo, aka latch2, aka staple latches) latches, Ib
    • latching which can be attempted post-incubation
    • can be incubated at 37°C for 1hr with the zipper oligos (c4.0.23, 24, 25), though latches can be used in the first incubation
  • inside-biotinylation w/o latches, Db
  • inside-biotinylation w/ latches, Hb

Stock ID	Experiment	1	2	3	4	4b	5	6	6b	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	total	H2O needed to 200uL
c4.0Eb	outside biotin -latches	66uL	33	33		4		4					4					2	2										148	52
c4.0Ib	outside biotin +latch design 2 (ie. 4-oligo)	66uL	33	33		4		4				4				2	2												148	52
c4.0Db	inside biotin -latches	66uL	33	33	4				4				4					2	2										148	52
c4.0Hb	inside biotin +latch design 2 (ie. 4-oligo)	66uL	33	33	4				4			4				2	2												148	52

• folded according to given protocol, using FOLDINGD

Folding for MgCl2 and 6x Titrations, Take Two

• because the gel from yesterday of the previoius titration showed no bands whatsoever, the reactions were refolded as before
• the reactions were, as usual, folded with the FOLDINGD program

3D models continued

Added scales to two images; Sketchup file now posted under Design 5 schematics.