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==Protocol-Part 5== <ol start="10"> <li>Column Purification-Qiaquick-Do this for 20 uL from Y and Z samples</li> <ol type="A"> <li>Add 100 uL (5X) PB (binding buffer) to 20 uL sample</li> <li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;</li> <li>Repeat wash step;</li> <li>Dry spin column for 1 minute 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>Elute with 30 uL EB</li> <li>Let stand 1 minute</li> <li>Spin for 1 minute at 14000 rpm</li></ol> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#808000;font-size:12pt" align="center" | width="95" height="30" | Sample Origin | width="95" | Sample New Letter |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Y | P |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | Z | Q |} ****** <li>Rolling Circle Amplification</li> '''Sample Conversion Table''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#808000;font-size:12pt" align="center" | width="95" height="30" | Sample Origin | width="95" | Sample New Letter | width="95" | Sample Numbers |- style="font-size:12pt" align="center" valign="bottom" | height="15" | X | F |1-5 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | Y | G |1,4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Z | H |1,4 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | P | I |1,4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Q | K |1,4 |} <ol type="A"> <li>Prepare 8.2X Master mix according to table below</li> <li>Add 12.5 uL master mix to each tube</li> <li>Add 4 uL template according to table</li> <li>Add 2.5 uL correct primer according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="250" height="30" | Reagent | width="85" | Single Rxn Vol | width="85" | 13.2X (A) MM uL | width="85" | 13.2X (B) MM uL |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 33 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | RCA Phosphothiorate Primer (10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 33 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 66 | align="center" align="center" valign="bottom" | 66 |- style="font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 26.4 | align="center" align="center" valign="bottom" | 26.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 13.2 | align="center" align="center" valign="bottom" | 13.2 |- style="font-size:12pt" | height="15" valign="bottom" | BSA (10 mg/mL) | align="center" align="center" valign="bottom" | 0.4 | align="center" align="center" valign="bottom" | 5.28 | align="center" align="center" valign="bottom" | 5.28 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 5.1 | align="center" align="center" valign="bottom" | 67.32 | align="center" align="center" valign="bottom" | 67.32 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 211.2 |style="font-weight:bold" align="center" align="center" valign="bottom" | 211.2 |} <li>Incubate at 95 C for 5 minutes</li> <li>Lower to 55C for 15 minutes</li> <li>Lower to 30C for at least 2 minutes</li> <li>Add 1 uL Phi29 polymerase to each sample</li> <li>Incubate 3 hours at 30C</li> <li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> </ol></ol> Continued [[Daniel:Notebook/ComboLock/2017-3-17|tomorrow]] [[Category:ComboLock]] [[Category:20170315]]
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