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class="far fa-moon"></i></div></div></nav><header class="d-flex justify-content-center align-items-center"><div class="container"><h1>RPA Lab Notebook</h1><p class="lead pl-1"></p><hr class="my-4"/></div></header><main><div class="container"><div class="row"><div class="sidebar col-lg-3"><div class="nav" id="contents"><h5>Contents</h5><ul></ul></div></div><div class="content col-lg-9"><article><h1>June 3rd, 2021</h1><h2>RPA Workflow</h2><p> **Done: ** -Preliminary temperature optimization (A2) →37ºC...2F, 10R →42ºC...4F, 11R</p></article></div></div></div></main><footer><div class="container"><p>Email: <a href="mailto:igem@oneonta.edu">iGEM@oneonta.edu</a> | <a href="https://suny.oneonta.edu/igem">suny.oneonta.edu/iGEM</a></p></div></footer><script src="https://2021.igem.org/Template:SUNY_Oneonta/content-bundleJS?action=raw&ctype=text/javascript"></script></body></html> | + | <!-- # TODO: #6 Fix table caption font--><!-- # TODO: #7 Fix citations links font size--><html lang="en"><head><meta charset="utf-8"/><meta content="width=device-width,initial-scale=1" name="viewport"/><title>RPA Lab Notebook | iGEM SUNY_Oneonta</title ><script src="https://2020.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML"></script><link href="https://2021.igem.org/Template:SUNY_Oneonta/css/contentCSS?action=raw&ctype=text/css" rel="stylesheet"/></head><body><!-- # TODO: #6 Fix table caption font--><!-- # TODO: #7 Fix citations links font size--><nav class="navbar navbar-expand-xl fixed-top"><div class="container d-flex justify-content-between"><a class="navbar-brand d-lg-inline-block" href="https://2021.igem.org/Team:SUNY_Oneonta"><h1>SNflaPs</h1></a><button aria-controls="navbarNav" aria-expanded="false" aria-label="Toggle navigation" class="navbar-toggler" data-target="#navbarNav" data-toggle="collapse" type="button"><span class="navbar-toggler-icon"></span></button><div class="collapse navbar-collapse" id="navbarNav"><ul class="navbar-nav 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class="far fa-moon"></i></div></div></nav><header class="d-flex justify-content-center align-items-center"><div class="container"><h1>RPA Lab Notebook</h1><p class="lead pl-1"></p><hr class="my-4"/></div></header><main><div class="container"><div class="row"><div class="sidebar col-lg-3"><div class="nav" id="contents"><h5>Contents</h5><ul></ul></div></div><div class="content col-lg-9"><article><h1>June 3rd, 2021</h1><h2>RPA Workflow</h2><p> <strong>Done: </strong></p><ul><li>Preliminary temperature optimization (A2) </li><li>37ºC...2F, 10R </li><li>42ºC...4F, 11R</ li></ul><p ><strong>To-Do:</strong></p><ol><li>Repeat temperature optimization (A2)</li><li>Optimize timing (20- 40 min)</li><li>Repeat primer + temperature + timing optimization (A2)</li><li>Try a real sample</li><li>Try with the heat block</li></ol><p>The TwistDX RPA protocols will be used for reaction experiment.</p><p><strong>Today:</strong> Made 10mL of Chloramphenicol stock aliquoted into 1000µL portions.</p><pre><code> Via updated “Antibiotic Stock Protocol” |
| | + | </code></pre><p><strong>In Wiki:</strong> Update “Antibiotic Stock Protocol – (1000 x 1 µL/mL Media) – 10mL Solutions”</p><p>Cm 1. Dissolve 0.25g of Chloramphenicol (25mg/mL) in reagent ethanol 10mL</p><ol start="2"><li>Store aliquots 1000µL into microcentrifuge tubes for storage at -20ºC</li></ol><p><strong>To Do:</strong></p><ul><li>Repeat experiment in Wiki RPA Figure 6 – Both primer pairs at both temperatures (40 min)</li><li>PCR Cleanup</li><li>Gel for product verification</li></ul><hr/><h1>June 7th, 2021</h1><h3>RPA Figure 6 Re-do</h3><div class="image"><img alt="Positive and Negative Controls at Different Temperatures" src="https://static.igem.org/mediawiki/2021/e/e2/T--SUNY_Oneonta--img--RPALN-Fig6Redo.png" style="width: 75%"/></div><p>The reaction will be set up using the protocol posted on the iGEM Teams Files.</p><table><thead><tr><th style="text-align:center">Reagent</th><th style="text-align:center">1 Reaction</th><th style="text-align:center">10 Reactions</th></tr></thead><tbody><tr><td style="text-align:center">2x Reaction Buffer</td><td style="text-align:center">25µL</td><td style="text-align:center">250µL</td></tr><tr><td style="text-align:center">10mM dNTPs</td><td style="text-align:center">1µL</td><td style="text-align:center">10µL</td></tr><tr><td style="text-align:center">Nuclease-free Water</td><td style="text-align:center">8.2µL</td><td style="text-align:center">82µL</td></tr><tr><td style="text-align:center">10x Probe E-mix</td><td style="text-align:center">5µL</td><td style="text-align:center">50µL</td></tr><tr><td colspan="3" style="text-align:center">--- (Add 20x Rxn mix to lid of Mastermix, invert 10 times and spin) ---</td></tr><tr><td style="text-align:center">20x Core Reaction Mix</td><td style="text-align:center">2.5µL</td><td style="text-align:center">25µL</td></tr></tbody></table><ul><li>2.5 µL of MgOAc to be added to each reaction.</li><li>1.0 µL of (DNA, water, or control DNA) to be added to each reaction.</li></ul><p><strong>PCR Cleanup:</strong></p><ul><li>50µL Product<ul><li>30µL elution buffer</li></ul></li></ul><p><strong>Gel:</strong> 0.4% agarose 50mL TBE 1x</p><ul><li>1kb marker</li><li>2F,10R samples: 5µL eluted product, 1µL of 6x Loading Dye</li><li>4F,11R samples: DILUTED: 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye</li></ul><hr/><h1>June 8th, 2021</h1></article></div></div></div></main><footer><div class="container"><p>Email: <a href="mailto:igem@oneonta.edu">iGEM@oneonta.edu</a> | <a href="https://suny.oneonta.edu/igem">suny.oneonta.edu/iGEM</a></p></div></footer><script src="https://2021.igem.org/Template:SUNY_Oneonta/content-bundleJS?action=raw&ctype=text/javascript"></script></body></html> |
