Daniel:Notebook/ComboLock/2016-7-21
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C Probe Test 3-SOD1/VIM-Stringent Washes (Started Monday)[edit]
Protocol[edit]
- Size Selection
- Combine 20 uL sample from each of the following sample lane: 1A, 1B, 2A, 2B, 3A
- 1A: human, normal, RVC hybridization buffer
- 2A: human, negative control, RVC hybridization buffer
- 1B: human, normal, normal hybridization buffer
- 2B: human, negative control, normal hybridization buffer
- 3B: mouse, normal, normal hybridization buffer
- Add 20 uL 6X loading dye
- Aliquot 30 uL each into 4 lanes
- 2 uL ladder with 5 uL gel loading dye and 23 uL TBE buffer
- Run gel for 20 min at 250V
- Stain with 3 uL SYBR Gold for 3 min
- Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- 2016-07-21-StringentWash-SizeSelect.png
Before Image
- 2016-07-21-StringentWash-SizeSelect-After.png
After Image
- Centrifuge gel at 14000rpm for 1.5 minutes
- Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
- Incubate at 37C for at least an hour with vigorous shaking
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer supernatant to a nanosep column
- Centrifuge at 14000rpm for 1.5 minutes
- Transfer flow through to a 2 mL tube
- Ethanol Precipitation
- Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
- Incubate overnight at -80C; continued tomorrow