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=RCA Test (Started [[Daniel:Notebook/ComboLock/2017-1-23|Monday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Epicentre Test (Started [[Daniel:Notebook/ComboLock/2017-1-25|Yesterday]])== '''Sample Matrix''' <ol start="2"> <li>qPCR</li> <ol type="A"> <li>Make 5.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>109.2 uL nfH2o</li> <li>5.2 uL AmpF</li> <li>130 uL 2X Kapa SYBR Fast Master Mix</li> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="100" height="45" | Sample | width="100" | Condition | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="85" | 2X Kapa SYBR qPCR MM | width="65" | 10 uM Forward Primer | width="65" | 10 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 6AC | align="center" | Epicentre | align="center" | A1 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 6BC | align="center" | Epicentre | align="center" | A2 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 6AD | align="center" | Epicentre 1:10 | align="center" | A3 | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 6BD | align="center" | Epicentre 1:10 | align="center" | A4 | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | | align="center" valign="bottom" | A5 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 50 |} <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> ****** <li>TBE Gel</li> <ol type="A"> <li>Mix 64 uL TBE, 16 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to 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> </ol></ol> ==Library Prep== Those bands are interesting to me. Time to sequence. '''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="160" height="30" | Sample | width="85" | Sample Amt | width="85" | Pre/Post RCA | width="85" | AmpR Index |- style="font-size:12pt" | height="15" valign="bottom" | Sample 6C (AB) | align="center" valign="bottom" | 1 amol | align="center" valign="bottom" | Epi | align="center" align="center" valign="bottom" | 22 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 6D (AB) | align="center" valign="bottom" | 1 amol | align="center" valign="bottom" | Epi 1:10 | align="center" align="center" valign="bottom" | 23 |} <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:2017-01-26-RCATest-Epicentre-SizeSelect.png|Before Image File:2017-01-26-RCATest-Epicentre-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/2017-1-27|tomorrow]]</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20170126-qPCR-RCATest-Epicentre.png|qPCR curve File:2017-01-26-RCATest-Epicentre.png|Gel image </gallery> ==Library Prep (Started [[Daniel:Notebook/ComboLock/2017-1-25|Yesterday]])== <ol start="2"> <li>Ethanol Precipitation</li> <ol type="A" start="3"> <li>Centrifuge for 30 minutes at 12500 rpm at 4C</li> <li>Remove supernatant and add 750 uL chilled 70% EtOH</li> <li>Centrifuge for 15 minutes at 12500 rpm at 4C</li> <li>Remove supernatant; dry in vacuum centrifuge for 15 minutes</li> <li>Resuspend in 20 uL H2O and consolidate samples</li> <li>Measure with Qbit</li> </ol></ol> [[Category:ComboLock]] [[Category:20170123]]
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