Daniel:Notebook/ComboLock/2016-8-20: Difference between revisions
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<li>Size Selection</li> | <li>Size Selection</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Add 20 uL sample (lanes 1-4), 5 uL 6X loading dye and 5 uL TBE to sample lanes</li> | |||
<li>Add 20 uL sample, 5 uL 6X loading dye and 5 uL TBE to sample lanes</li> | |||
<li>Aliquot 30 uL each into 4 sample lanes</li> | <li>Aliquot 30 uL each into 4 sample lanes</li> | ||
<li>2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer</li> | <li>2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer</li> |
Revision as of 20:58, 20 August 2016
Positive Control Amplicon Test (Started Thursday)
Protocol
- 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
- 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