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=Padlock0301 Test (Started [[Daniel:Notebook/ComboLock/2016-10-7|Friday 10-7]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] Repeating the qPCR performed on [[Daniel:Notebook/ComboLock/2016-10-8|Saturday]]. That run failed. I learned later that I had made an error in the amplification primer (I was using 2 reverse primers). I designed and ordered AmpF-CL-v2, which should be correct. ==Protocol== <ol start="6"> <li>qPCR</li> <ol type="A"> <li>Set up reaction according to table in next section</li> <li>Make master mixes according to following recipes</li> <ol type="a"> <li>264.25 uL nfH2O</li> <li>377.5 uL SYBR Fast</li> <li>7.55 uL 100 uM AmpF-CL-v2</li> </ol> <li>Add 43 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 x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 144 uL TBE, 36 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> ===qPCR Sample Table=== {| 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="100" height="45" | Sample | width="65" | Template Molecule Count | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF-CL-v2 | width="65" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | align="center" valign="bottom" | 10 pmol | align="center" valign="bottom" | A1 | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | align="center" valign="bottom" | 10 pmol | align="center" valign="bottom" | B1 | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | align="center" valign="bottom" | 10 pmol | align="center" valign="bottom" | C1 | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2 | align="center" valign="bottom" | None | align="center" valign="bottom" | D1 | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 3 | align="center" valign="bottom" | 10 pmol | align="center" valign="bottom" | E1 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4A | align="center" valign="bottom" | 100 fmol | align="center" valign="bottom" | F1 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4B | align="center" valign="bottom" | 100 fmol | align="center" valign="bottom" | G1 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4C | align="center" valign="bottom" | 100 fmol | align="center" valign="bottom" | H1 | align="center" align="center" valign="bottom" | 23 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 5A | align="center" valign="bottom" | 1 fmol | align="center" valign="bottom" | A12 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 5B | align="center" valign="bottom" | 1 fmol | align="center" valign="bottom" | B12 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 5C | align="center" valign="bottom" | 1 fmol | align="center" valign="bottom" | C12 | align="center" align="center" valign="bottom" | 24 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 6A | align="center" valign="bottom" | 10 amol | align="center" valign="bottom" | D12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 6B | align="center" valign="bottom" | 10 amol | align="center" valign="bottom" | E12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 6C | align="center" valign="bottom" | 10 amol | align="center" valign="bottom" | F12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | G12 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 19.5 | align="center" align="center" valign="bottom" | 50 |} ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161011-qPCR-Padlock0301-AmpFCLv2.png|qPCR Curve File:2016-10-11-Padlock0301-AmpFCLv2-Gel2.png|Gel Image 1 File:2016-10-11-Padlock0301-AmpFCLv2-Gel1.png|Gel Image 2 </gallery> [[Category:ComboLock]] [[Category:20161007]]
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