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=Antibodies Test (Started [[Daniel:Notebook/ComboLock/2017-6-14|Wednesday 6/14]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Library Prep== <ol> <li>Qiaquick Purification</li> <ol type="A"> <li>Combine samples into a 1.5 mL tube following matrix</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>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 40 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 20 uL sample and 4 uL 6X dye per sample</li> <li>Mix 16 uL TE, 4 uL ladder, and 4 uL 6X dye per sample</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>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 heights=200px widths=200px mode=packed-hover> File:2017-06-19-AntibodyTest-CS-SizeSelect.png|Pre Selection File:2017-06-19-AntibodyTest-CS-SizeSelect-After.png|Post Selection </gallery> <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/2017-6-20|tomorrow]] [[Category:ComboLock]] [[Category:20170614]]
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