Daniel:Notebook/ComboLock/2016-8-25
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Positive Control Amplicon Test 2 (Started Monday)
Protocol-Library Prep
- Size Selection
- Add 20 uL sample (lanes 1-4), 5 uL 6X loading dye and 5 uL TBE to sample lanes
- Aliquot 30 uL each into 4 sample lanes
- 2 uL ladder with 5 uL gel loading dye and 28 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
- 2016-08-20-PosConTest-SizeSelect.png
Before Image
- 2016-08-20-PosConTest-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 Monday