IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-8
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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 (TBD based on further experiments below)
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. (Fifth centrifuge step for 3.5 min)
- 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 (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
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