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=Positive Control Dilution Test (Started [[Daniel:Notebook/ComboLock/2016-8-26|Friday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== Today I'm testing the samples using the P2 and P12RC amplification primers. Yesterday included the original amplicon materials; this qPCR should be only the DNA still attached to beads. <ol start=6> <li>qPCR X2-Primers</li> <ol type="A"> <li>Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC</li> <li>Load lanes according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B7DEE8;font-size:12pt;font-weight:bold" align="center" | width="105" height="31" | Sample Name | width="105" | Sample No. | width="105" | qPCR Lane |- style="font-size:12pt" align="center" | height="15" | Pure C1-C2 | Sample 00 | A1 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 1E+11 | Sample 0 | B1 |- style="font-size:12pt" align="center" | align="center" height="15" | 1E+10 | Sample 1 | C1 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 1E+09 | Sample 2 | D1 |- style="font-size:12pt" align="center" | align="center" height="15" | 1E+08 | Sample 3 | E1 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 1E+07 | Sample 4 | F1 |- style="font-size:12pt" align="center" | align="center" height="15" | 1E+06 | Sample 5 | G1 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 1E+05 | Sample 6 | H1 |- style="font-size:12pt" align="center" | align="center" height="15" | 10000 | Sample 7 | A12 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 1000 | Sample 8 | B12 |- style="font-size:12pt" align="center" | align="center" height="15" | 100 | Sample 9 | C12 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 10 | Sample 10 | D12 |- style="font-size:12pt" align="center" | align="center" height="15" | 1 | Sample 11 | E12 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | height="15" | NTC | NTC | F12 |} <li>Master Mix</li> <ol type="a"> <li>352.5 uL KAPA SyberFast MM</li> <li>310.2 uL nfH2O</li> <li>7.05 uL primer2</li> <li>7.05 uL primer12RC</li></ol> <li>Add 48 uL Master Mix to each well</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 x32</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube</li> <li>Add 1 or 2 uL sample according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#E6B8B7;font-size:12pt;font-weight:bold" align="center" | width="85" height="31" | Gel Lane | width="106" | Sample Name | width="85" | qPCR Lane | width="85" | uL Sample |- style="font-size:12pt" align="center" | align="center" height="15" | 2 | 25 bp Ladder | align="center" | | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 3 | 1E11 Molecule | B1 | align="center" | 1 |- style="font-size:12pt" align="center" | align="center" height="15" | 4 | 1E9 Molecule | D1 | align="center" | 1 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 5 | 1E7Molecule | F1 | align="center" | 2 |- style="font-size:12pt" align="center" | align="center" height="15" | 6 | 1E5 Molecule | H1 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 7 | 1000 Molecule | B12 | align="center" | 2 |- style="font-size:12pt" align="center" | align="center" height="15" | 8 | 10 Molecule | D12 | align="center" | 2 |- style="background-color:#D9D9D9;font-size:12pt" align="center" | align="center" height="15" | 9 | 1 Molecule | E12 | align="center" | 2 |} <li>Add 10 uL mix to well</li> <li>Run gel for 25 minutes at 250V</li> <li>Stain for 3 min with 3 uL SYBR gold</li> <li>Image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20160829-X2Dilution-Primers-qPCR.png|qPCR Curves File:2016-08-29-X2Dilution-PrimerAmpGel.png|Representative Gel Image </gallery>
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