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==Library Prep== Based on [[Daniel:Notebook/ComboLock/2017-1-24|yesterday]]'s results, I'm going to sequence the most successful lanes (PreRCA-1pmol; PostRCA-1 pmol, 10 fmol, and 100 amol). With the UMI now on the padlock, this should give a relative percentage of padlock capture. Each sample has a unique AmpR index. '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | width="120" height="32" | Sample | width="90" | Sample Amt | width="70" | Pre/Post RCA | width="90" | AmpR Index |- style="font-size:12pt" | height="15" valign="bottom" | Sample 0 (AB) | align="center" valign="bottom" | 1 pmol | align="center" valign="bottom" | Pre | align="center" align="center" valign="bottom" | 21 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 0X (AB) | align="center" valign="bottom" | 1 pmol | align="center" valign="bottom" | Post | align="center" align="center" valign="bottom" | 25 |- style="font-size:12pt" | height="15" valign="bottom" | Sample 2X (AB) | align="center" valign="bottom" | 10 fmol | align="center" valign="bottom" | Post | align="center" align="center" valign="bottom" | 26 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4X (AB) | align="center" valign="bottom" | 100 amol | align="center" valign="bottom" | Post | align="center" align="center" valign="bottom" | 27 |} <ol> <li>Size Select Gel</li> <ol type="A"> <li>Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)</li> <li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li> <li>Aliquot 48/24 uL per sample/ladder lane into each well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> <li>Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li> <li>Image in gel doc</li> <gallery perrow=2 heights=300px widths=300px> File:2017-01-25-RCATest-SizeSelect.png|Before Image File:2017-01-25-RCATest-SizeSelect-After.png|After Image </gallery> <li>Centrifuge at 12000 rpm for 1.5 minutes</li> <li>Add 500 uL TE to each sample</li> <li>Incubate 4 hours at 37C with vigorous shaking;</li> <li>Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom</li> <li>Extract supernatant and add to nanosep column</li> <li>Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant</li></ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample</li> <li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2017-1-26|tomorrow]]</li> </ol></ol> [[Category:ComboLock]] [[Category:20170123]]
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