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==Positive Control Test== [[Daniel:Notebook/ComboLock/2017-2-13|Yesterday]]'s results showed that the positive controls failed time and time again. Looking at the math again, by adding 2 uL 10 uM template to the positive control reactions, I was adding >100X the amount Kapa's protocol recommends. So this time I ran a dilution series that covers each of the positive controls in dilutions of undiluted to 1:10000. <ol> <li>qPCR</li> <ol type="A"> <li>Make qPCR master mixes according to following recipes</li> '''Master Mix Recipes''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | width="65" height="45" | Master Mix | width="119" | Primer Pair | width="65" | Master Mix NoX | width="65" | Forward Primer Amt (10 uM) | width="65" | Reverse Primer Amt (10 uM) | width="65" | uL nfH2O | width="65" | uL 2X Kapa SYBR Fast | width="65" | Total |- style="font-size:12pt" | height="15" valign="bottom" | MMT | align="center" valign="bottom" | PCCT-F/PCCT-R | align="center" align="center" | 11.2 | align="center" align="center" | 11.2 | align="center" align="center" | 11.2 | align="center" align="center" | 235.2 | align="center" align="center" | 280 |style="font-weight:bold" align="center" align="center" | 537.6 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | MMC1 | align="center" valign="bottom" | primer24/primer4RC | align="center" align="center" | 11.2 | align="center" align="center" | 11.2 | align="center" align="center" | 11.2 | align="center" align="center" | 235.2 | align="center" align="center" | 280 |style="font-weight:bold" align="center" align="center" | 537.6 |- style="font-size:12pt" | height="15" valign="bottom" | MMC2 | align="center" valign="bottom" | primer6/primer12RC | align="center" align="center" | 11.2 | align="center" align="center" | 11.2 | align="center" align="center" | 11.2 | align="center" align="center" | 235.2 | align="center" align="center" | 280 |style="font-weight:bold" align="center" align="center" | 537.6 |} <li>Add 48 uL of appropriate master mix and 2 uL sample according to plate layout</li> <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 x45</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol></ol> ===Plate Layout=== [[Image:PlateLayout-20170214.png|600px]] '''Note''': Blank indicates lanes that were measured but had nothing (even master mix) in them.
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