Daniel:Notebook/ComboLock/2016-8-25: Difference between revisions

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>Djacobse
>Djacobse
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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 lanes 1 and 8-10, 5 uL 6X loading dye and 5 uL TBE to sample lanes</li>
<ol type="a">
<li>Lane 1: X2-1X</li>
<li>Lane 8: X3-50X</li>
<li>Lane 9: X2-Flip</li>
<li>Lane 10:X3-Flip</li></ol>
<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 23:23, 25 August 2016

Positive Control Amplicon Test 2 (Started Monday)

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Protocol-Library Prep

  1. Size Selection
    1. Add 20 uL sample lanes 1 and 8-10, 5 uL 6X loading dye and 5 uL TBE to sample lanes
      1. Lane 1: X2-1X
      2. Lane 8: X3-50X
      3. Lane 9: X2-Flip
      4. Lane 10:X3-Flip
    2. Aliquot 30 uL each into 4 sample lanes
    3. 2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer
    4. Run gel for 20 min at 250V
    5. Stain with 3 uL SYBR Gold for 3 min
    6. 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
    7. Centrifuge gel at 14000rpm for 1.5 minutes
    8. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
    9. Incubate at 37C for at least an hour with vigorous shaking
    10. Centrifuge at 14000rpm for 1.5 minutes
    11. Transfer supernatant to a nanosep column
    12. Centrifuge at 14000rpm for 1.5 minutes
    13. 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 Monday