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)