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==Protocol-Part 5== <ol start="8"> <li>qPCR</li> <ol type="A"> <li>Make 17.2X master mix according to following (1X)</li> <ol type="a"> <li>361.2 uL nfH<sub>2</sub>O (21)</li> <li>17.2 uL 10 uM AmpF6.4Sol (1)</li> <li>430 uL 2X Kapa SYBR Fast Master Mix (25)</li></ol> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout</li> [[Image:PlateLayout-20170501-ProductionRun-PostBglii.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>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> <gallery perrow=3 heights=300px widths=300px> File:20170501-qpCR-ProductionRun-PostBglii.png|qPCR curves (NEB amplified samples blue; TF amplified samples red) File:2017-05-01-ProductionRun-PostBglii-NEB-NoAmp.png|NEB Gel File:2017-05-01-ProductionRun-PostBglii-TF-NoAmp.png|Thermo Fisher RCA File:2017-05-01-ProductionRun-PostBglii-NEB-PostAmp.png|NEB Gel-Post qPCR File:2017-05-01-ProductionRun-PostBglii-TF-PostAmp.png|ThermoFisher Gel Post qPCR </gallery> Although the gel quality is poor, I do like these results, especially the pre-qPCR bands in the ThermoFisher lanes. I'm going to check the results via sequencing. See [[Daniel:Notebook/ComboLock/2017-5-2|tomorrow]]. [[Category:ComboLock]] [[Category:20170428]]
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