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=Circular Product Production (Started [[Daniel:Notebook/ComboLock/2017-3-22|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol> <li>qPCR</li> <ol type="A"> <li>Make qPCR master mix according to following recipes</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CC66FF;font-size:12pt;font-weight:bold" align="center" | width="250" height="36" | Reagent | width="80" | 1X Rxn | width="120" | MM-Amp (6.2X) | width="120" | MM-Prod (7.2X) |- style="font-size:12pt" | height="15" valign="bottom" | Primer Pair | align="center" valign="bottom" | NA | align="center" valign="bottom" | AmpF/AmpR | align="center" valign="bottom" | p24/p12RC |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Forward Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 7.2 |- style="font-size:12pt" | height="15" valign="bottom" | Forward Primer (10 uM) | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 7.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Kapa SYBR Fast Master Mix (2X) | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 155 | align="center" align="center" valign="bottom" | 180 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 130.2 | align="center" align="center" valign="bottom" | 151.2 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 48 |style="font-weight:bold" align="center" align="center" valign="bottom" | 297.6 |style="font-weight:bold" align="center" align="center" valign="bottom" | 345.6 |} <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170323-CPProduction.png|600px]] <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 x30</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>Qubit Measurement</li> <ol type="A"> <li>Add 995 uL buffer and 5 uL ssdye to tube</li> <li>Add 190 uL buffer and 10 uL correct standard to standard tubes</li> <li>Add 199 uL buffer and 1 uL sample to sample tubes</li> <li>Let sit 5-10 minutes in the dark</li> <li>Measure in Qubit</li></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 120 uL TBE and 40 uL 6X dye</li> <li>Aliquot 10 uL per sample on to parafilm</li> <li>Add 2 uL sample or 1.5 uL ladder to aliquots</li> <li>Mix and add 10 uL per lane to gel</li> <li>Run gel at 230V for 24 minutes</li> <li>Add 2 uL SYBR gold and incubate on shaker for 3 minutes</li> <li>Rinse and image in gel doc</li></ol> </ol> ===Results=== <gallery perrow=3 heights=300px widths=300px> File:20170323-qPCR-CPProd.png|qPCR curves File:2017-03-23-CPProd-qPCR-CPTarget.png|Circular Product target (104bp) File:2017-03-23-CPProd-qPCR-AmpF-AmpR.png|AmpF/AmpR (221 bp) </gallery> Use the AmpF/AmpR gel as a reference point in the future. Also, positive controls for qPCR and the like should be done at a 1:10 dilution of the original. The original concentration, via Qubit is: {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#80FF00;font-size:12pt;font-weight:bold" align="center" | width="85" height="30" | Qubit | width="85" | Dil | width="105" | Meas | width="105" | Amt | width="105" | Average (ng/uL) | width="105" | uM |- style="font-size:12pt" | height="15" valign="bottom" | x1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 118 | align="center" align="center" valign="bottom" | 118 | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | x2 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 18.3 | align="center" align="center" valign="bottom" | 91.5 | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |- style="font-size:12pt" | height="15" valign="bottom" | x3 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 8.94 | align="center" align="center" valign="bottom" | 89.4 | align="center" align="center" valign="bottom" | 99.6 | align="center" align="center" valign="bottom" | 1.61 |}
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