Daniel:Notebook/ComboLock/2017-6-19: Difference between revisions

From ZhangLabWiki
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 20 uL each sample into a 1.5 mL tube following matrix</li>
<li>Combine samples into a 1.5 mL tube following matrix</li>


<li>Add 200 uL (5X) PB (binding buffer) to sample</li>
{| 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>Let stand with cover open in fume hood for ~5 minutes</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 30 uL nfH<sub>2</sub>O to column</li>
<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 30 uL 10X TBE and 24 uL 6X dye</li>
<li>Mix 20 uL sample and 4 uL 6X dye per sample</li>
<li>Aliquot 9 uL for each sample/ladder into a separate tube</li>
<li>Mix 16 uL TE, 4 uL ladder, and 4 uL 6X dye per sample</li>
<li>Add 15 uL sample or 2.5/12.5 uL ladder/10X TBE to aliquot tubes</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)

Back to Calendar

Library Prep

  1. Qiaquick Purification
    1. Combine samples into a 1.5 mL tube following matrix
    2. 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
    3. Add 600 uL (5X) PB (binding buffer) to sample
    4. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    5. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    6. Repeat above step with 400 uL PE buffer
    7. Dry spin column for 2 minutes at 14000 rpm; discard flow through
    8. Transfer column to a new 1.5 mL eppendorf tube
    9. Add 40 uL TE to column
    10. Spin for 1 minute at 14000 rpm
  2. Size Select TBE Gel
    1. Mix 20 uL sample and 4 uL 6X dye per sample
    2. Mix 16 uL TE, 4 uL ladder, and 4 uL 6X dye per sample
    3. Aliquot 24 uL for each sample/ladder into a separate tube
    4. Mix and add 23 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc
    8. Cut out bands at approximately 221bp
    9. Add extracted gel bands to 0.5mL tube with 0.2 gauge needle hole inside a 1.5 mL tube
    10. Centrifuge for 1:30 at 13000rpm
    11. Throw away 0.5mL tube and add 500 uL TE buffer to each tube
    12. Incubate at 37C for 1 hour
    13. Centrifuge for 1:30 at 13000rpm
    14. Withdraw supernatant and add it to a nanosep column
    15. Centrifuge for 1:30 at 13000 rpm
    16. 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