Daniel:Notebook/ComboLock/2016-8-17: Difference between revisions

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>Djacobse
>Djacobse
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<li>Shred the gel by centrifuging at 14000rpm for 1 min 30 sec</li>
<li>Shred the gel by centrifuging at 14000rpm for 1 min 30 sec</li>
<li>Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking</li>
<li>Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking</li>
<li>Centrifuge at 12000rpm for 1.5 min</li>
<li>Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm</li>
</ol>
<li>Ethanol Precipitation</li>
<ol type="A">
<li>Move flow through to a 2 mL tube</li>
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue</li>
<li>Incubate at -80C for 4 hours</li>
<li>Pellet by centrifuging for 25 min at 12000rpm at 4C</li>
<li>Remove supernatant and add 750 uL chilled 70% EtOH</li>
<li>Centrifuge for 12 min at 12000 rpm and 4 C</li>
<li>Remove supernatant and air dry</li>
<li>Resuspend in 30 uL nfH2O and measure in nanodrop</li>
</ol>


[[Category:ComboLock]] [[Category:20160805]]
[[Category:ComboLock]] [[Category:20160805]]

Revision as of 22:16, 17 August 2016

Positive Control Amplicon Production (Started yesterday)

Back to Calendar

Protocol

  1. Size select gel
    1. Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
    2. Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
    3. Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
    4. Run the gel at 250V for 25 min
    5. Stain with 3 uL SYBR gold for 3 min
    6. Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
    7. Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
    8. Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
    9. Centrifuge at 12000rpm for 1.5 min
    10. Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
  2. Ethanol Precipitation
    1. Move flow through to a 2 mL tube
    2. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
    3. Incubate at -80C for 4 hours
    4. Pellet by centrifuging for 25 min at 12000rpm at 4C
    5. Remove supernatant and add 750 uL chilled 70% EtOH
    6. Centrifuge for 12 min at 12000 rpm and 4 C
    7. Remove supernatant and air dry
    8. Resuspend in 30 uL nfH2O and measure in nanodrop