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==Protcol== <ol start="2"> <li>Latch and Padlock Hybridization</li> <ol type="A" start="5"> <li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li> </ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 uL exonuclease III (100 units/μL) in 1:1 ratio</li> <li>Add 2 μL of exonuclease I/III mix to reaction</li> <li>Mix the reaction by swirling pipette around the well 5 times</li> <li>Incubate reaction at 37 ºC for 2 hours</li> <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> </ol> <li>qPCR</li> <ol type="A"> <li>Set up reaction according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | width="85" height="45" | Sample | width="65" | Lane | width="65" | Batch | width="65" | AmpR Index | width="65" | Cells | width="105" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AmpF 6.4 | width="65" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 | align="center" valign="bottom" | A1 | align="center" valign="bottom" | B1 | align="center" align="center" valign="bottom" | 1 | 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:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 2 | align="center" valign="bottom" | A2 | align="center" valign="bottom" | B2 | align="center" align="center" valign="bottom" | 2 | 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" | height="15" valign="bottom" | Sample 3 | align="center" valign="bottom" | A3 | align="center" valign="bottom" | B3 | align="center" align="center" valign="bottom" | 3 | 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:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" valign="bottom" | A4 | align="center" valign="bottom" | B4 | align="center" align="center" valign="bottom" | 4 | 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" | height="15" valign="bottom" | Sample 5 | align="center" valign="bottom" | A5 | align="center" valign="bottom" | B5 | align="center" align="center" valign="bottom" | 5 | 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:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 6 | align="center" valign="bottom" | A6 | align="center" valign="bottom" | B6 | align="center" align="center" valign="bottom" | 6 | 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" | height="15" valign="bottom" | Sample 7 | align="center" valign="bottom" | A7 | align="center" valign="bottom" | B7 | align="center" align="center" valign="bottom" | 7 | 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 |} <li>Master Mix</li> <ol type="a"> <li>165 uL KAPA SyberFast MM</li> <li>115.5 uL nfH2O</li> <li>3.3 uL AmpF 6.4</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 x20</li> <li>72C 2 min</li> <li>16C hold</li> </ol> </ol> ===qPCR Results=== <gallery perrow=2 heights=200px widths=200px> File:VIM-Ab-LPTest2 Results 20160628.png|qPCR Curves File:2016-06-29-CombolockAb-LP-Test2.jpg|Gel image </gallery> The results are confusing since even the negative control samples amplified. I need to repeat the experiment.
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