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==Random Hexamer RCA== RCA Test (again) using random hexamers. <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 4.5X 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="200" height="30" | Reagent | width="65" | uL Added | width="90" | Master Mix (4.5X) |- 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" | Random Hexamer (100 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 11.25 |- 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" | 3.6 |- 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" | 9 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 4.5 |- 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" | 39.15 |- 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" | 67.5 |} <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> <li>Qubit Measure</li> <ol type="A"> <li>Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)</li> <li>Add 190 uL dye/buffer and 10 uL standard to standard tubes</li> <li>Add 199 uL dye/buffer and 1 uL sample to sample tubes</li> <li>Wait 5 minutes to allow dye saturation</li> <li>Measure in Qubit</li> </ol></ol> ===Results=== {| 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="130" height="36" | Sample Name | width="65" | 1A | width="65" | 1B | width="85" | 1A6 | width="85" | 1B6 | width="85" | 4A6 | width="85" | 4B6 |- style="font-size:12pt" | height="30" | Sample Condition | align="center" | Sample-PreRCA | align="center" | Sample-PreRCA | align="center" | Sample | align="center" | Sample | align="center" | No PO4 (PCCB) | align="center" | No PO4 (PCCB) |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | RCA Primer | align="center" | None | align="center" | None | align="center" | Random Hexamer | align="center" | Random Hexamer | align="center" | Random Hexamer | align="center" | Random Hexamer |- style="font-size:12pt" | height="15" | Conc (ng/uL) | align="center" align="center" | 0.81 | align="center" align="center" | 1.27 | align="center" align="center" | 16.6 | align="center" align="center" | 16.5 | align="center" align="center" | 15.4 | align="center" align="center" | 20.5 |- style="font-size:12pt" | height="30" valign="bottom" | Fold Change from Pre-RCA | align="center" | NA | align="center" | NA | align="center" align="center" | 20.5 | align="center" align="center" | 13.0 | align="center" align="center" | 19.0 | align="center" align="center" | 16.1 |} So either everything amplified (weakly) or nothing amplified. I think it's everything weakly.
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