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

1. Centrifuged at 14k g for 2.5 min, removed 200+ μL flow-through, and added 200 μL water.
2. Repeated previous step four more times, placing each flow-through in different 0.5 mL PCR tubes. (Fifth centrifuge step for 3.5 min)
3. Vacufuged flow-through to near dryness

Trial 2: 40 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)

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 (____uL of flowthrough obtained)
  • removed the flowthrough to separate 1.5mL tube
  • added ____uL of H2O to top
  • repeated twice more
  • extracted final ____uL by turning reservoir upside down and spinning at 1000g for 1 min

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)

• 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