IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-20: Difference between revisions
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== Folding 5.0.A and 6.0 == | == Folding 5.0.A and 6.0 == | ||
=Design 5= | |||
*Make working stock c5.0.A | *Make working stock c5.0.A | ||
{| {{table}} | {| {{table}} | ||
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|} | |} | ||
=Design 6= | |||
* Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5) | * Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5) | ||
** To mix pre-working stocks from plates, pipet 10 {{ul}} of each of the appropriate oligos into 1.5 mL tubes. | ** To mix pre-working stocks from plates, pipet 10 {{ul}} of each of the appropriate oligos into 1.5 mL tubes. | ||
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|c6.0C||out aptamers||1 (153 {{ul}}), 3 (6 {{ul}}), 4 (29 {{ul}})||12 {{ul}}||200 {{ul}} | |c6.0C||out aptamers||1 (153 {{ul}}), 3 (6 {{ul}}), 4 (29 {{ul}})||12 {{ul}}||200 {{ul}} | ||
|} | |} | ||
= Folding protocol = | |||
* Need to figure out concentration of p7308 | |||
== PEG precipitation == | == PEG precipitation == | ||
Revision as of 09:51, 20 August 2006
Folding 5.0.A and 6.0
Design 5
- Make working stock c5.0.A
| Working Stocks | Description | Pre-Working Stocks | Water | Total |
| c5.0A | no latches, no aptamers | 1 (86 μL), 2 (49 μL), 3 (49 μL), 4 (2.5 μL), 5 (2.5 μL), 6 (1.5 μL), 7 (1.5 μL) | 8 μL | 200 μL |
Design 6
- Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5)
- To mix pre-working stocks from plates, pipet 10 μL of each of the appropriate oligos into 1.5 mL tubes.
- Mix working stocks c6.0.A, c6.0.B, c6.0.C
Pre-Working Stocks
| Description | Plate Locations | Total |
| c6.0.1: core barrel oligos | box_v6_A; box_v6_B rows A-D (1-12), row E (1-9) | 153 |
| c6.0.2: barrel oligos @ outside aptamers -aptamers | box_v6_B row E (9-12), row F (1-12) | 15 |
| c6.0.3: barrel oligos @ inside aptamers -aptamers | box_v6_B row G (1-6) | 6 |
| c6.0.4: barrel oligos @ outside aptamers+ligand | box_v6_B row G (7-12), row H (1-12); box_v6_C row A (1-11) | 29 |
| c6.0.5: barrel oligos @ inside aptamers+ligand | box_v6_C row A (12), row B (1-12), row C (1-5) | 18 |
Working Stock
| Stock ID | Experiment | 1 | 2 | 3 | 4 | 5 |
| c6.0A | no aptamers | x | x | x | - | - |
| c6.0B | in aptamers | x | x | - | - | x |
| c6.0C | out aptamers | x | - | x | x | - |
| Working Stocks | Description | Pre-Working Stocks | Water | Total |
| c6.0A | no aptamers | 1 (153 μL), 2 (15 μL), 3 (6 μL) | 26 μL | 200 μL |
| c6.0B | in aptamers | 1 (153 μL), 2 (15 μL), 5 (18 μL) | 14 μL | 200 μL |
| c6.0C | out aptamers | 1 (153 μL), 3 (6 μL), 4 (29 μL) | 12 μL | 200 μL |
Folding protocol
- Need to figure out concentration of p7308
PEG precipitation
- Goal: to get cleaner purification of oligos away from nanostructures and to increase the volume of purified nanostructures we have for protection assays.
- Nanostructures:
- using 30mM MgCl2, 1x oligos (seems to work best given previous results).
- designs: c5.0.A, c5.0.C, c5.0.D key
- PEG: 8%, 10%. Total volume in each is 100 μL
- 8 % Cocktail:
40 μL 20% PEG
10 μL 5M NaCl
10 μL water
40 μL Nanostructures (add last)
- 10 % Cocktail:
50 μL 20% PEG
10 μL 5M NaCl
40 μL Nanostructures (add last)
- incubate on ice for 15 min.
- spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
- carefully pipette off supernatant
- resuspended "pellet" in 50 μL of water
- note: add PEG first, nanostructures last; mix using tapping after everything added. let sit for ~5 min. before putting it on ice
