IGEM:Harvard/2006/vlau/Notebook/2006-8-2
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Folding Rxn
1. Goal: 6 samples of 100 μL 6 hb
2. Reaction mixture:
50μL p7308 scaffold 40μL oligos 10μL 10x folding buffer (500mM HEPES ph 7.5, 500mM NaCl, 100mM MgCl2) neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water
3. Image (2% agarose gel + 11 mM MgCl2)
Lane | Contents | Loading Dye |
1 | 1 kb Ladder (4 μL) | - |
2 | folded 6 hb (4 μL) | 2 μL |
3 | negative control: no scaffold (4 μL) | 2 μL |
4 | negative control: no oligos (4 μL) | 2 μL |
PEG Precipitation
1. Goal: repeat titration of PEG precipitation conditions for 2 samples of 6hb
2. Protocol
Sample | % PEG | 20% PEG Volume | 5 M NaCl Volume | dH2O | Total Volume |
6hb: 40 μL | 3% | 30 μL | 20 μL | 110 μL | 200 μL |
6hb: 40 μL | 4% | 40 μL | 20 μL | 100 μL | 200 μL |
6hb: 40 μL | 5% | 50 μL | 20 μL | 90 μL | 200 μL |
6hb: 40 μL | 10% | 100 μL | 20 μL | 40 μL | 200 μL |
6hb: 40 μL | 14% | 140 μL | 20 μL | 0 μL | 200 μL |
- incubated on ice for 15 min. - spun @ 16k rcf for 10 min. - pipetted supernatant into separate tube - pellet resuspended in 1x folding buffer volume equal to supernatant - ran equivalent volumes of pellet and supernatant on 2% agarose gel (+11 mM MgCl2)
3. Gel Analysis
Lane | Contents | Loading Dye |
1 | 1 kb Ladder (4 μL) | - |
2 | untreated 6hb (10 μL) | 2 μL |
3 | 3% supernatant (10 μL) | 2 μL |
4 | 3% pellet (10 μL) | 2 μL |
5 | 4% supernatant (10 μL) | 2 μL |
6 | 4% pellet (10 μL) | 2 μL |
7 | 5% supernatant (10 μL) | 2 μL |
8 | 5% pellet (10 μL) | 2 μL |
9 | 10% supernatant (10 μL) | 2 μL |
10 | 10% pellet (10 μL) | 2 μL |
11 | 14% supernatant (10 μL) | 2 μL |
12 | 14% pellet (10 μL) | 2 μL |