IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1: Difference between revisions
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* goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold | * goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold | ||
* methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with [http://probes.invitrogen.com/media/pis/mp11494.pdf Invitrogen SYBR | * methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with [http://probes.invitrogen.com/media/pis/mp11494.pdf Invitrogen SYBR Gold]. | ||
** prepared 1x solution: 10 {{ul}} thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4{{c}}. | ** prepared 1x solution: 10 {{ul}} thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4{{c}}. | ||
Revision as of 09:43, 1 August 2006
To do today
- ordering
- new oligo ligands
- AscIII (if not already ordered
- PEG precipitations
- repeat experiment, run on PA gel, run on agarose gel
- SYBR gold
- read technical specs
- determine lower threshold of imaging
SYBR gold testing
- goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold
- methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with Invitrogen SYBR Gold.
- prepared 1x solution: 10 μL thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4[[:Category:{{{1}}}|{{{1}}}]].
| lane | DNA (3.2.7.2b) (12.5 pg DNA / fmol) | ||
| (μL) | (fmols) | (pg) | |
| 1 | 15 μL 1 μM | 15,000 | 187,500 |
| 2 | 10 μL 1 μM | 10,000 | 125,000 |
| 3 | 5 μL 1 μM | 5,000 | 62,500 |
| 4 | 2.5 μL 1.0 μM | 2,500 | 31,250 |
| 5 | 1 μL 1.0 μM | 1,000 | 12,500 |
| 6 | 5 μL 0.1 μM | 500 | 6,250 |
| 7 | 2.5 μL 0.1 μM | 250 | 3,125 |
| 8 | 1 μL 0.1 μM | 100 | 1,250 |
| 9 | 5 μL 0.01 μM | 50 | 625 |
| 10 | 2.5 μL 0.01 μM | 25 | 313 |
| 11 | 1 μL 0.01 μM | 10 | 125 |
| 12 | 2 μL 1kb+ ladder | ||
PEG precipitation
- Goal: repeat titration of PEG precipitation conditions for folded containers
- Folding reactions
- 2 samples of 6hb (100 μL final volume)
- 4 samples of design 5 barrels (100 μL volume)
- Mix the following
- 10 uL 500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2
- 40 uL working stock 250 nM each oligo (100 nM each oligo final concentration)
- 50 uL p7308 20 nM (10 nM final concentration)
- Anneal from 80[[:Category:{{{1}}}|{{{1}}}]] to 20[[:Category:{{{1}}}|{{{1}}}]], -1[[:Category:{{{1}}}|{{{1}}}]] per min
- PEG precipitations
- Plan to use 200 μL final volume
- Trying 4%, 6%, 8%, 10%, 12%, 14%
- Add 40 μL 10 nM scaffold/100 nM oligo folded mix
- Add 20% PEG solution (40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL
- Add 5 M NaCl stock solution (20 μL)
- Gel Analysis
- Analyze on 2% Mg agarose gel (0.5x TBE, 11 mM MgCl2, 0.5 μg/mL EtBr)
- Load 1kb ladder (1 lane)
- Load c5 supernatant, pellet for 0%, 4%, 6%, 8%, 10%, 12%, 14% (13 lanes)
- Load 6hb supernatant, pellet for 0%, 4%, 14% (5 lanes)
- 19 lanes total
