IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-8: Difference between revisions
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# Repeated previous step four more times, placing each flow-through in different 0.5 mL PCR tubes. | # Repeated previous step four more times, placing each flow-through in different 0.5 mL PCR tubes. | ||
Trial 2: 40 {{ul}} folded nanostructures diluted in 160 {{ul}} water (total volume: 200 {{ul | Trial 2: 40 {{ul}} folded nanostructures diluted in 160 {{ul}} water (total volume: 200 {{ul}}) and placed in a Microcon YM-50 tube. Centrifuged as follows, removing (and combining) flow-through at given intervals: | ||
{| {{table}} | {| {{table}} |
Revision as of 13:42, 8 August 2006
Folding Rxns w/ New Ligands
Working stocks
- c3.2F (latch 1, in aptamers)
- c3.2G (latch 1, out aptamers)
- c3.2H (latch 2, in aptamers)
- c3.2I (latch 2, out aptamers)
- c4.0F (latch 1, in atpamers)
- c4.0G (latch 1, out aptamers)
- c4.0H (latch 2, in aptamers)
- c4.0I (latch 2, out aptamers)
- c5.0C (latch 2, in aptamers)
- c5.0D (latch 2, out aptamers)
- 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
Digestion
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.
- 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.
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 appears nearly dry) |