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===Expansion PCR=== 1. Set up 8 reactions for each of the 8 probes (64 reactions total) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="right" width="149" height="16" | |style="font-weight:bold" width="65" | per rxn |style="font-weight:bold" width="65" | V4S1 |style="font-weight:bold" width="65" | V4S2 |style="font-weight:bold" width="65" | V4S3 |style="font-weight:bold" width="65" | V4S4 |- style="font-size:12pt" align="center" | height="16" | 20 nM OligoPool | align="right" | 0.2 | align="right" | 1.7 | align="right" | 1.7 | align="right" | 1.7 | align="right" | 1.7 |- style="font-size:12pt" align="center" | height="16" | 2X Kapa SYBR qPCR MM | align="right" | 50 | align="right" | 425 | align="right" | 425 | align="right" | 425 | align="right" | 425 |- style="font-size:12pt" align="center" | height="16" | 100 uM AP1V41U | align="right" | 0.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 |- style="font-size:12pt" align="center" | height="16" | 100 uM AP2V4 | align="right" | 0.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 |- style="font-size:12pt" align="center" | height="16" | 2 mM aa-dUTP | align="right" | 3 | align="right" | 25.5 | align="right" | 25.5 | align="right" | 25.5 | align="right" | 25.5 |- style="font-size:12pt" align="center" | height="16" | H20 | align="right" | 46 | align="right" | 391 | align="right" | 391 | align="right" | 391 | align="right" | 391 |- style="font-size:12pt" align="center" | height="16" | Total Volume | align="right" | 100 | align="right" | 850 | align="right" | 850 | align="right" | 850 | align="right" | 850 |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="right" height="16" | |style="font-weight:bold" | per rxn |style="font-weight:bold" | V6S1 |style="font-weight:bold" | V6S2 |style="font-weight:bold" | V6S3 |style="font-weight:bold" | V6S4 |- style="font-size:12pt" align="center" | height="16" | 20 nM OligoPool | align="right" | 0.2 | align="right" | 1.7 | align="right" | 1.7 | align="right" | 1.7 | align="right" | 1.7 |- style="font-size:12pt" align="center" | height="16" | 2X Kapa SYBR qPCR MM | align="right" | 50 | align="right" | 425 | align="right" | 425 | align="right" | 425 | align="right" | 425 |- style="font-size:12pt" align="center" | height="16" | 100 uM AP1V61U | align="right" | 0.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 |- style="font-size:12pt" align="center" | height="16" | 100 uM AP2V6 | align="right" | 0.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 | align="right" | 3.4 |- style="font-size:12pt" align="center" | height="16" | 2 mM aa-dUTP | align="right" | 3 | align="right" | 25.5 | align="right" | 25.5 | align="right" | 25.5 | align="right" | 25.5 |- style="font-size:12pt" align="center" | height="16" | H20 | align="right" | 46 | align="right" | 391 | align="right" | 391 | align="right" | 391 | align="right" | 391 |- style="font-size:12pt" align="center" | height="16" | Total Volume | align="right" | 100 | align="right" | 850 | align="right" | 850 | align="right" | 850 | align="right" | 850 |} 2. qPCR 95 C 30 seconds Repeat 16x 95 C 3 seconds 55 C 30 seconds 72 C 20 seconds Read Plate 72 C 2 min 15 C Hold stopped qPCR for all samples except V4S1 and V4S2 at cycle 12 stopped qPCR for V4S1 and V4S2 at final cycle
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