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==RCA Repeat== Gonna try RCA again, since the adapter testing above showed product with at least the adapter regions. That means either the bridge is present or the circular product itself. So repeating RCA should increase the concentration of product, if available. <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 8.5X Master mix according to table below</li> <li>Add 15 uL master mix to each tube</li> <li>Add sample to new reaction tubes according to following 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="160" height="30" | Reagent | width="65" | uL Added | width="90" | Master Mix (8.5X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (LinkerL-RevComp; 10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 21.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 6.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Phi29 Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.7 | align="center" align="center" valign="bottom" | 73.95 |- 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" | 127.5 |} <li>Incubate at 37C for 3 hours</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> Continued [[Daniel:Notebook/ComboLock/2017-1-19|tomorrow]] [[Category:ComboLock]] [[Category:20170113]]
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