Daniel:Notebook/ComboLock/2017-6-19: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 8: | Line 8: | ||
<li>Qiaquick Purification</li> | <li>Qiaquick Purification</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Combine | <li>Combine samples into a 1.5 mL tube following matrix</li> | ||
<li>Add | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#FF6666;font-size:12pt;font-weight:bold" align="center" | |||
| width="135" height="33" | Tube | |||
| width="95" | uL Added | |||
| width="95" | Index | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1A | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" align="center" valign="bottom" | 30 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1B | |||
| align="center" align="center" valign="bottom" | 50 | |||
| align="center" align="center" valign="bottom" | 31 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 (AB) | |||
| align="center" valign="bottom" | 50 (ea) | |||
| align="center" align="center" valign="bottom" | 32,33 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 (AB) | |||
| align="center" valign="bottom" | 50 (ea) | |||
| align="center" align="center" valign="bottom" | 34,35 | |||
|} | |||
<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>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>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>Repeat above step with 400 uL PE buffer</li> | ||
<li>Dry spin column for 2 minutes at 14000 rpm; discard flow through</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>Transfer column to a new 1.5 mL eppendorf tube</li> | ||
<li>Add | <li>Add 40 uL TE to column</li> | ||
<li>Spin for 1 minute at 14000 rpm</li> | <li>Spin for 1 minute at 14000 rpm</li> | ||
</ol> | </ol> | ||
<li>Size Select TBE Gel</li> | <li>Size Select TBE Gel</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 20 uL sample and 4 uL 6X dye per sample</li> | ||
<li> | <li>Mix 16 uL TE, 4 uL ladder, and 4 uL 6X dye per sample</li> | ||
<li> | <li>Aliquot 24 uL for each sample/ladder into a separate tube</li> | ||
<li>Mix and add 23 uL per lane to gel</li> | <li>Mix and add 23 uL per lane to gel</li> | ||
<li>Run gel at 230V for 24 minutes</li> | <li>Run gel at 230V for 24 minutes</li> |
Revision as of 19:56, 19 June 2017
Production Run (Started Wednesday 5/8)
Library Prep
- Qiaquick Purification
- Combine samples into a 1.5 mL tube following matrix
- 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 40 uL TE to column
- Spin for 1 minute at 14000 rpm
- Size Select TBE Gel
- Mix 20 uL sample and 4 uL 6X dye per sample
- Mix 16 uL TE, 4 uL ladder, and 4 uL 6X dye per sample
- Aliquot 24 uL for each sample/ladder into a separate tube
- Mix and add 23 uL per lane to gel
- 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
- 2017-06-19-AntibodyTest-CS-SizeSelect.png
Pre Selection
- 2017-06-19-AntibodyTest-CS-SizeSelect-After.png
Post Selection
- 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
Tube | uL Added | Index |
Sample 1A | 50 | 30 |
Sample 1B | 50 | 31 |
Sample 2 (AB) | 50 (ea) | 32,33 |
Sample 3 (AB) | 50 (ea) | 34,35 |
Continued tomorrow