IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-8: Difference between revisions
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| 5.5||177 (but Microcon appears nearly dry) | | 5.5||177 (but Microcon appears nearly dry) | ||
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==Folding c3.2, c4.0, and c6.0 Reactions== | |||
* according to usual protocols, folded: | |||
<pre> | |||
1 rxn of: | |||
c3.2F | |||
c3.2G | |||
c3.2H | |||
c3.2I | |||
c4.0F | |||
c4.0G | |||
c4.0H | |||
c4.0I | |||
--- | |||
2 rxns of: | |||
c6.0A | |||
c6.0B | |||
c6.0C | |||
</pre> | |||
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* mixed and folded 8 40uL reactions of each according to usual protocol and with FOLDINGD program | * mixed and folded 8 40uL reactions of each according to usual protocol and with FOLDINGD program | ||
==Microcon of c3.2F, G, H, and I== | |||
* after assessing the success of the Microcon 1-spin method, we will use Microcon tubes to purify four c3.2 designs: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" colspan="3" style="background:#f0f0f0;"|core | |||
| align="center" colspan="4" style="background:#f0f0f0;"|aptamers | |||
| align="center" colspan="9" style="background:#f0f0f0;"|latches | |||
| align="center" colspan="2" style="background:#f0f0f0;"|latch displacement | |||
|- | |||
| align="center" style="background:#f0f0f0;"|'''Stock ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Experiment''' | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''3''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| align="center" style="background:#f0f0f0;"|'''6o''' | |||
| align="center" style="background:#f0f0f0;"|'''7o''' | |||
| align="center" style="background:#f0f0f0;"|'''8''' | |||
| align="center" style="background:#f0f0f0;"|'''9''' | |||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
| align="center" style="background:#f0f0f0;"|'''11''' | |||
| align="center" style="background:#f0f0f0;"|'''12''' | |||
| align="center" style="background:#f0f0f0;"|'''13''' | |||
| align="center" style="background:#f0f0f0;"|'''14''' | |||
| align="center" style="background:#f0f0f0;"|'''15''' | |||
| align="center" style="background:#f0f0f0;"|'''16''' | |||
| align="center" style="background:#f0f0f0;"|'''17''' | |||
| align="center" style="background:#f0f0f0;"|'''18''' | |||
|- | |||
| c3.2.Fo || +latch1 +attachment-ligand-site-IN ||||x||x||x|| ||x||x|| || || ||x||x|| ||x||x|| || || || | |||
|- | |||
| c3.2.Go || +latch1 +attachment-ligand-site-OUT ||||x||x||x||x|| || ||x|| || ||x||x|| ||x||x|| || || || | |||
|- | |||
| c3.2.Ho || +latch2 +attachment-ligand-site-IN ||||x||x||x|| ||x||x|| || || ||x|| ||x||x|| ||x||x|| || | |||
|- | |||
| c3.2.Io || +latch2 +attachment-ligand-site-OUT ||||x||x||x||x|| || ||x|| || ||x|| ||x||x|| ||x||x|| || | |||
|} |
Revision as of 15:34, 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 (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) |
Folding c3.2, c4.0, and c6.0 Reactions
- according to usual protocols, folded:
1 rxn of: c3.2F c3.2G c3.2H c3.2I c4.0F c4.0G c4.0H c4.0I --- 2 rxns of: c6.0A c6.0B c6.0C
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 | - | - | - | - | - | - | - | - | - | - |
c5.0E(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
Microcon of c3.2F, G, H, and I
- after assessing the success of the Microcon 1-spin method, we will use Microcon tubes to purify four c3.2 designs:
core | aptamers | latches | latch displacement | |||||||||||||||||
Stock ID | Experiment | 1 | 2 | 3 | 4 | 5 | 6o | 7o | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | |
c3.2.Fo | +latch1 +attachment-ligand-site-IN | x | x | x | x | x | x | x | x | x | ||||||||||
c3.2.Go | +latch1 +attachment-ligand-site-OUT | x | x | x | x | x | x | x | x | x | ||||||||||
c3.2.Ho | +latch2 +attachment-ligand-site-IN | x | x | x | x | x | x | x | x | x | x | |||||||||
c3.2.Io | +latch2 +attachment-ligand-site-OUT | x | x | x | x | x | x | x | x | x | x |