IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-4: Difference between revisions
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*Plan to use 100 {{ul}} final volume | *Plan to use 100 {{ul}} final volume | ||
*Add 20 {{ul}} 10 nM scaffold/100 nM oligo folded mix | *Add 20 {{ul}} 10 nM scaffold/100 nM oligo folded mix | ||
*Add 20% PEG | *Add 20% PEG/2.5M NaCl solution (20 {{ul}}) | ||
*Add as much water to each as it takes to get them to a 100 {{ul}} final volume (60 {{ul}}) | |||
*Add as much water to each as it takes to get them to a 100 {{ul}} final volume ( | |||
*Incubate on ice for 15 minutes | *Incubate on ice for 15 minutes | ||
*Spin 16k rcf for 10 minutes | *Spin 16k rcf for 10 minutes |
Revision as of 10:52, 4 August 2006
PAGE of Concentrated Eb, Fb, and Gb
- given amounts of streptavidin were added to each component on parafilm
- 2uL of 10x loading dye was added to each mixture subsequently
- gel was run at 4°C for 1hr 10minutes at 100V
- gel was stained in coomassie blue from 12PM F 8/4/06 to ________
lane | component | concentration | amount of component in well μL | streptavidin added μL |
1 | streptavidin | 2uM | 3.5 | - |
2 | p7308 | 44uM | 7.5 | 3.5 |
3 | c4.0.6b (ie. biotinylated inside-oligos) | 50nM each oligo (as in all pre-working stocks) | 7 | 3.5 |
4 | Eb PEG-pellet | 21/8 of a rxn | 15 | 3.5 |
5 | Fb PEG-pellet | 21/8 of a rxn | 15 | 3.5 |
6 | Gb PEG-pellet | 21/8 of a rxn | 15 | 3.5 |
7 | streptavidin | 2uM | 7 | - |
PEG precipitation w/ New Folded Rxns
- Goal: test PEG precipitation with folding rxns under new conditions
- Plan to use 100 μL final volume
- Add 20 μL 10 nM scaffold/100 nM oligo folded mix
- Add 20% PEG/2.5M NaCl solution (20 μL)
- Add as much water to each as it takes to get them to a 100 μL final volume (60 μL)
- Incubate on ice for 15 minutes
- Spin 16k rcf for 10 minutes
- Pipette out supernatant into separate tube
- Resuspend pellet in 1x folding buffer volume equal to the supernatant
- Run equivalent volumes of pellet and sup on 2% agarose gel (+ 11 mM MgCl2)
- 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 naked scaffold (1 lane)
- Load 6 folding rxns untreated, supernatant, pellet (18 lanes)
- 20 lanes total
Lane | Contents | Loading Dye |
0 | 1kb DNA ladder (4 μL) | 0 μL |
1 | naked scaffold (10 μL) | 2 μL |
2 | 10mM MgCl2 + 100nM oligos rxn untreated (10 μL) | 2 μL |
3 | 10mM MgCl2 + 100nM oligos rxn pellet (10 μL) | 2 μL |
4 | 10mM MgCl2 + 100nM oligos rxn supernatant (10 μL) | 2 μL |
5 | 20mM MgCl2 + 100nM oligos rxn untreated (10 μL) | 2 μL |
6 | 20mM MgCl2 + 100nM oligos rxn pellet (10 μL) | 2 μL |
7 | 20mM MgCl2 + 100nM oligos rxn supernatant (10 μL) | 2 μL |
8 | 30mM MgCl2 + 100nM oligos rxn untreated (10 μL) | 2 μL |
9 | 30mM MgCl2 + 100nM oligos rxn pellet (10 μL) | 2 μL |
10 | 30mM MgCl2 + 100nM oligos rxn supernatant (10 μL) | 2 μL |
11 | 10mM MgCl2 + 600nM oligos rxn untreated (10 μL) | 2 μL |
12 | 10mM MgCl2 + 600nM oligos rxn pellet (10 μL) | 2 μL |
13 | 10mM MgCl2 + 600nM oligos rxn supernatant (10 μL) | 2 μL |
14 | 20mM MgCl2 + 600nM oligos rxn untreated (10 μL) | 2 μL |
15 | 20mM MgCl2 + 600nM oligos rxn pellet (10 μL) | 2 μL |
16 | 20mM MgCl2 + 600nM oligos rxn supernatant (10 μL) | 2 μL |
17 | 30mM MgCl2 + 600nM oligos rxn untreated (10 μL) | 2 μL |
18 | 30mM MgCl2 + 600nM oligos rxn pellet (10 μL) | 2 μL |
19 | 30mM MgCl2 + 600nM oligos rxn supernatant (10 μL) | 2 μL |