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

Folding Rxns w/ New Ligands

Working stocks

Design 3

• c3.2F (latch 1, in aptamers)
• c3.2G (latch 1, out aptamers)
• c3.2H (latch 2, in aptamers)
• c3.2I (latch 2, out aptamers)

Design 4

• c4.0F (latch 1, in atpamers)
• c4.0G (latch 1, out aptamers)
• c4.0H (latch 2, in aptamers)
• c4.0I (latch 2, out aptamers)

Design 5

• c5.0C (latch 2, in aptamers)
• c5.0D (latch 2, out aptamers)

Design 6

• c6.0A (no aptamers)
• c6.0B (in aptamers)
• c6.0C (out aptamers)

Folding protocol

Each reaction:

• 9 μL p7308 scaffold (44 nM)
• 16 μL oligo stock (250 nM)
• 4 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl₂)
• 11 μL dH₂O

Folding conditions:

• start at 80[[:Category:{{{1}}}|{{{1}}}]], decrement 1[[:Category:{{{1}}}|{{{1}}}]] every 2 min. for 60 cycles.

Incubations

• to each reaction, add 1 μL 5 μM oligo ligand and titurate
• incubate at room temperature for 5 min.
  • final concentration of oligo ligands: ~500 nM (50-fold excess of scaffold, 17-fold or 13-fold excess of nanostructure-bound attachment oligos, 1.7-fold or 1.3-fold excess of all attachment oligos)
• to each reaction, add 2 μL 2 μM latch mixture and titurate
• incubate at room temperature for 5 min.
  • final concentration of latch oligos: ~100 nM (10-fold excess of scaffold)

Purification

...with Microcon tube protocol (TBD based on further experiments below)

Gel Analysis

• Run 2% agarose gel, supplemented to 10 mM MgCl₂
• Run in 1x TBE supplemented to 10 mM MgCl₂

Microcon trials

Trial 1: 30 μL folded nanostructures diluted in 360 μL water and placed in a Microcon YM-50 tube.

• Centrifuged at 14k g for 2.5 min, removed 200+ μL flow-through, and added 200 μL water.
• Repeated previous step four more times, placing each flow-through in different 0.5 mL PCR tubes. (Fifth centrifuge step for 3.5 min)
• Centrigued remaining liquid out by turning tube upside down and centrifuging at 1.3k g for 1 min (manual says 1k g for 3 min., so we should've centrifuged longer)
• Vacufuged flow-through to near dryness

Trial 2: 30 μL folded nanostructures diluted in 160 μL water (total volume: 200 μL) and placed in a Microcon YM-50 tube. Centrifuged as follows, removing (and combining) flow-through at given intervals:

Cumulative centrifuge time (min.) at 14k g	Cumulative flow-through (μL)
2.5	120
3.5	150
4.5	170
5.5	177 (but Microcon appears nearly dry)

• Centrigued remaining liquid out by turning tube upside down and centrifuging at 1.3k g for 1 min (manual says 1k g for 3 min., so we should've centrifuged longer)
• Vacufuged flow-through to near dryness

Gel analysis

2% agarose gel with EtBr, both gel and buffer (1x TBE) supplemented to 10 mM MgCl₂. Run at 60V for 1 h.

• 

  2% agarose, EtBr, 1hr @ 60V

Lane	Contents
1	1 kb+ ladder
2	p7308
3	4.0.H (30 μL)
4	Trial 1 retained
5	Trial 1 flow-through 1
6	Trial 1 flow-through 2
7	Trial 1 flow-through 3
8	Trial 1 flow-through 4
9	Trial 1 flow-through 5
10	Trial 2 retained
11	Trial 2 flow-through

Biotinylated c5.0

• reconstituted the oligos c5.0.8.1-5 and c5.0.9.1-5 (in tubes) to 200uM in EB
• made pre-working stocks c5.0.8 and c5.0.9
  • 2.5uL of each 200uM oligo solution + 7.5uL of dH2O
• made working stocks c5.0 E(b) and c5.0 F(b):

Stock ID	Experiment	1	2	3	4	4L	5	5L	6	7	8(b)	9(b)	10+11	12	13+14	15	16+17	18	19	20	21
c5.0E(b)	no latches, outside biotin	172uL	-	-	-	-	5	-	3	3	5	-	-	-	-	-	-	-	-	-	-
c5.0F(b)	no latches, intside biotin	172uL	-	-	5	-	-	-	3	3	-	5	-	-	-	-	-	-	-	-	-

• mixed and folded 8 40uL reactions of each according to usual protocol and with FOLDINGD program
• Microconned all 8x40uL (=320uL) of Eb and Fb:
  • spun the 320uL of reaction for 4.5min (190uL of flowthrough obtained)
  • removed the flowthrough to separate 1.5mL tube
  • added 190uL of H2O to top
  • repeated twice more
  • spun down once for an additional 1min, without adding extra H2O
  • extracted final 30uL by turning reservoir upside down and spinning at 1000g for 1 min
  • b/c the amount of "elute" was so low (under 10uL) after this step, despite the flowthrough being 30uL short of the 200uL of liquid that was placed in the top, steps for "spinning to dryness" - though the top membrane never appeared completely dry - were taken as outlined by Microcon:
    • 30uL of H2O were added to the reservoir membrane
    • the reservoir and vial were gently agitated (setting 3 on the Vortex) for 30 seconds
    • reservoir was flipped, placed into the collection tube again, and spun down again at 1000g for 3min

Microcon of c3.2F, G, H, and I

• after assessing the success of the Microcon 1-spin method, we've decided to use Microcon tubes to purify the four c3.2 designs given above (working stock table).
• diluted each 40uL sample to 200uL and then spin down for 4.5minutes at 14000g
  • remove flowthrough to separate 1.5mL tube
  • add 170ul of H20 to top
  • repeat twice more (ie. three flowthrough fractions)
• flip the reservoir and place in new clean Microncon vial; spin down at 1000g for 1min
  • b/c the amount of "elute" was so low (under 10uL) after this step, despite the flowthrough being 30uL short of the 200uL of liquid that was placed in the top, steps for "spinning to dryness" - though the top membrane never appeared completely dry - were taken as outlined by Microcon:
    • 30uL of H2O were added to the reservoir membrane
    • the reservoir and vial were gently agitated (setting 3 on the Vortex) for 30 seconds
    • reservoir was flipped, placed into the collection tube again, and spun down again at 1000g for 3min

• we will run the top liquid and the flowthroughs in a Mg2+ 2% agarose gel (ladder, scaffold, 4 top liquids - 10uL of each, 4 flowthroughs - Speedvac for 30 minutes down to ~40uL = 10 lanes)

DNA Nano and Perry's Work - because we rock

• see Perry's page