Daniel:Notebook/ComboLock/2017-7-10: Difference between revisions

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==
=Oligo+Protein Test (Started [[Daniel:Notebook/ComboLock/2017-7-6|Thursday July 6]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]
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==Library Prep==
==Library Prep==
<ol>
<li>Qiaquick Purification</li>
<ol type="A">
<li>Combine samples A+B for each condition into a 1.5 mL tube</li>
<li>Add 600 uL (5X) PB (binding buffer) to sample</li>
<li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li>
<li>Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through</li>
<li>Repeat above step with 400 uL PE buffer</li>
<li>Dry spin column for 2 minutes at 14000 rpm; discard flow through</li>
<li>Transfer column to a new 1.5 mL eppendorf tube</li>
<li>Add 50 uL TE to column</li>
<li>Spin for 1 minute at 14000 rpm</li>
</ol>
<li>Size Select TBE Gel</li>
<ol type="A">
<li>Mix 30 uL sample and 6 uL 6X dye per sample</li>
<li>Mix 26 uL TE, 4 uL ladder, and 6 uL 6X dye per sample</li>
<li>Aliquot 36 uL for each into a separate lane</li>
<li>Run gel at 230V for 24 minutes</li>
<li>Add 2 uL SYBR gold and incubate on shaker for 3 minutes</li>
<li>Rinse and image in gel doc</li>
<li>Cut out bands at approximately 221bp</li>


<gallery perrow=2 mode=packed-hover heights=250px widths=250px>
<gallery perrow=2 mode=packed-hover heights=250px widths=250px>
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File:2017-07-10-Oligo-and-Ab-SizeSelect-After.png|After
File:2017-07-10-Oligo-and-Ab-SizeSelect-After.png|After
</gallery>
</gallery>
*Note that this is a pretty bad gel, so I had to take wider fractions than I would have liked. We'll see how the sequencing results look
<li>Add extracted gel bands to 0.5mL tube with 0.2 gauge needle hole inside a 1.5 mL tube</li>
<li>Centrifuge for 1:30 at 13000rpm</li>
<li>Throw away 0.5mL tube and add 500 uL TE buffer to each tube</li>
<li>Incubate at 37C for 1 hour</li>
<li>Centrifuge for 1:30 at 13000rpm</li>
<li>Withdraw supernatant and add it to a nanosep column</li>
<li>Centrifuge for 1:30 at 13000 rpm</li>
<li>Remove flow through and add it to new 2 mL tube (~500 uL)</li>
</ol>
<li>Ethanol Precipitation</li>
<ol type="A">
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1 uL glycoblue to each tube</li>
<li>Incubate overnight at -80C</li>
</ol>
</ol>
Continued [[Daniel:Notebook/ComboLock/2017-7-11|tomorrow]]
[[Category:ComboLock]] [[Category:20170706]]

Latest revision as of 19:44, 10 July 2017

Oligo+Protein Test (Started Thursday July 6)[edit]

Back to Calendar


Library Prep[edit]

  1. Qiaquick Purification
    1. Combine samples A+B for each condition into a 1.5 mL tube
    2. Add 600 uL (5X) PB (binding buffer) to sample
    3. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    4. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    5. Repeat above step with 400 uL PE buffer
    6. Dry spin column for 2 minutes at 14000 rpm; discard flow through
    7. Transfer column to a new 1.5 mL eppendorf tube
    8. Add 50 uL TE to column
    9. Spin for 1 minute at 14000 rpm
  2. Size Select TBE Gel
    1. Mix 30 uL sample and 6 uL 6X dye per sample
    2. Mix 26 uL TE, 4 uL ladder, and 6 uL 6X dye per sample
    3. Aliquot 36 uL for each into a separate lane
    4. Run gel at 230V for 24 minutes
    5. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    6. Rinse and image in gel doc
    7. Cut out bands at approximately 221bp
      • Note that this is a pretty bad gel, so I had to take wider fractions than I would have liked. We'll see how the sequencing results look
    8. Add extracted gel bands to 0.5mL tube with 0.2 gauge needle hole inside a 1.5 mL tube
    9. Centrifuge for 1:30 at 13000rpm
    10. Throw away 0.5mL tube and add 500 uL TE buffer to each tube
    11. Incubate at 37C for 1 hour
    12. Centrifuge for 1:30 at 13000rpm
    13. Withdraw supernatant and add it to a nanosep column
    14. Centrifuge for 1:30 at 13000 rpm
    15. Remove flow through and add it to new 2 mL tube (~500 uL)
  3. Ethanol Precipitation
    1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1 uL glycoblue to each tube
    2. Incubate overnight at -80C

Continued tomorrow