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=Cell Trial Padlock0201 (Started [[Daniel:Notebook/ComboLock/2016-10-17|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="3"> <li>Circularization</li> <ol type="A"> <li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li></ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Prepare exonuclease I/III mix by mixing 7.5 uL exonuclease I (20 units/μL) and 1.5 uL exonuclease III (100 units/μL) in 1:1 ratio</li> <li>Add 2 μL of exonuclease I/III mix to reaction</li> <li>Mix the reaction by swirling pipette around the well 5 times</li> <li>Incubate reaction at 37 ºC for 2 hours</li> <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> </ol> <li>qPCR</li> <ol type="A"> <li>Prepare the following master mix</li> <ol type="a"> <li>89.25 uL nfH2O</li> <li>2.55 uL 100 uM AmpF6.4Sol</li> <li>127.5 uL Kapa SYBR Fast MM</li></ol> <li>Add 43 uL master mix to each well</li> <li>Add sample and AmpR indexes according to sample table below</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="105" height="45" | Sample | width="120" | Condition | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="100" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF6.4Sol | width="74" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="82" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1 | align="center" | Exp. Rep 1 | align="center" | A1 | align="center" align="center" | 20 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.5 | align="center" align="center" | 5 | align="center" align="center" | 17.5 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 2 | align="center" | Exp. Rep 2 | align="center" | B1 | align="center" align="center" | 20 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.5 | align="center" align="center" | 5 | align="center" align="center" | 17.5 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3 | align="center" | No C Probes | align="center" | C1 | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.5 | align="center" align="center" | 5 | align="center" align="center" | 17.5 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4 | align="center" | No Cells | align="center" | D1 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 0.5 | align="center" align="center" | 5 | align="center" align="center" | 17.5 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" | No qPCR sample | align="center" | E1 | align="center" align="center" | 23 | align="center" align="center" | 0 | align="center" align="center" | 25 | align="center" align="center" | 0.5 | align="center" align="center" | 5 | align="center" align="center" | 19.5 | align="center" align="center" | 50 |} <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 x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 56 uL TBE, 14 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161018-qPCR-CellTestPadlock0201.png|qPCR Curve File:2016-10-18-CellTest-Padlock0201.png|Gel Image </gallery>
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