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]] | ||
Line 5: | Line 5: | ||
==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]
Library Prep[edit]
- Qiaquick Purification
- Combine samples A+B for each condition into a 1.5 mL tube
- Add 600 uL (5X) PB (binding buffer) to sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Repeat above step with 400 uL PE buffer
- Dry spin column for 2 minutes at 14000 rpm; discard flow through
- Transfer column to a new 1.5 mL eppendorf tube
- Add 50 uL TE to column
- Spin for 1 minute at 14000 rpm
- Size Select TBE Gel
- Mix 30 uL sample and 6 uL 6X dye per sample
- Mix 26 uL TE, 4 uL ladder, and 6 uL 6X dye per sample
- Aliquot 36 uL for each into a separate lane
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
- Cut out bands at approximately 221bp
- 2017-07-10-Oligo-and-Ab-SizeSelect.png
Before
- 2017-07-10-Oligo-and-Ab-SizeSelect-After.png
After
- 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
- Add extracted gel bands to 0.5mL tube with 0.2 gauge needle hole inside a 1.5 mL tube
- Centrifuge for 1:30 at 13000rpm
- Throw away 0.5mL tube and add 500 uL TE buffer to each tube
- Incubate at 37C for 1 hour
- Centrifuge for 1:30 at 13000rpm
- Withdraw supernatant and add it to a nanosep column
- Centrifuge for 1:30 at 13000 rpm
- Remove flow through and add it to new 2 mL tube (~500 uL)
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1 uL glycoblue to each tube
- Incubate overnight at -80C
Continued tomorrow