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==RCA Test== Since it seems like its RCA that is the problem I will do RCA again, this time testing both AmpF and AmpR as (dirty) primers. To save on sample space, I'll use 1 and 4 again (4 is a suitable negative control). I should know whether or not the product amplifies in even half an hour, so we're gonna go with that (measure using Qubit) <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 (AmpF or AmpRInd21; 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 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>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="91" height="24" | Sample Name | width="60" | 1A | width="76" | 1B | width="71" | 1AY | width="65" | 1BY | width="65" | 1AF | width="65" | 1BF | width="65" | 4AF | width="65" | 4BF | width="65" | 1AR | width="65" | 1BR | width="65" | 4AR | width="65" | 4BR |- style="font-size:12pt" | height="45" | Sample Condition | align="center" | Sample-PreRCA | align="center" | Sample-PreRCA | align="center" | Sample | align="center" | Sample | align="center" | Sample | align="center" | Sample | align="center" | No Phosphate (PCCB) | align="center" | No Phosphate (PCCB) | align="center" | Sample | align="center" | Sample | align="center" | No Phosphate (PCCB) | align="center" | No Phosphate (PCCB) |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | RCA Primer | align="center" | None | align="center" | None | align="center" | LLRC (Mine) | align="center" | LLRC (Mine) | align="center" | AmpF | align="center" | AmpF | align="center" | AmpF | align="center" | AmpF | align="center" | AmpR | align="center" | AmpR | align="center" | AmpR | align="center" | AmpR |- 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" | 0.72 | align="center" align="center" | 0.62 | align="center" align="center" | 35.1 | align="center" align="center" | 46.9 | align="center" align="center" | 35 | align="center" align="center" | 40.7 | align="center" align="center" | 25.3 | align="center" align="center" | 26.8 | align="center" align="center" | 31.5 | align="center" align="center" | 30.2 |} ===Discussion (RCA Test)=== From the results on the table, I'd say there was amplification (on the order of 35X for sample 1) with AmpF '''and''' AmpR. Based on the potential dirtiness of the AmpF and AmpR primers, I'm going to repeat the experiment tomorrow using my adapter primers. Continued [[Daniel:Notebook/ComboLock/2017-1-20|tomorrow]] [[Category:ComboLock]] [[Category:20170113]]
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