Daniel:Notebook/ComboLock/2016-8-20: Difference between revisions

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(Created page with "=Positive Control Amplicon Test (Started Thursday)= Back to Calendar ==Protocol== <ol start="6"> <li>S...")
 
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<li>Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample</li>
<li>Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample</li>
<li>Incubate overnight at -80C; continued [[Daniel:Notebook/ComboLock/2016-7-22|tomorrow]]</li></ol>
<li>Incubate overnight at -80C; continued [[Daniel:Notebook/ComboLock/2016-8-21|tomorrow]]</li></ol>
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Revision as of 20:55, 20 August 2016

Positive Control Amplicon Test (Started Thursday)

Back to Calendar

Protocol

  1. Size Selection
    1. Combine 20 uL sample from each of the following sample lane: 1A, 1B, 2A, 2B, 3A
      1. 1A: human, normal, RVC hybridization buffer
      2. 2A: human, negative control, RVC hybridization buffer
      3. 1B: human, normal, normal hybridization buffer
      4. 2B: human, negative control, normal hybridization buffer
      5. 3B: mouse, normal, normal hybridization buffer
    2. Add 20 uL sample, 5 uL 6X loading dye and 5 uL TBE to sample lanes
    3. Aliquot 30 uL each into 4 sample lanes
    4. 2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer
    5. Run gel for 20 min at 250V
    6. Stain with 3 uL SYBR Gold for 3 min
    7. 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
    8. Centrifuge gel at 14000rpm for 1.5 minutes
    9. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
    10. Incubate at 37C for at least an hour with vigorous shaking
    11. Centrifuge at 14000rpm for 1.5 minutes
    12. Transfer supernatant to a nanosep column
    13. Centrifuge at 14000rpm for 1.5 minutes
    14. Transfer flow through to a 2 mL tube
  2. Ethanol Precipitation
    1. Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
    2. Incubate overnight at -80C; continued tomorrow