− | <!-- # 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 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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” | + | <!-- # 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 ml-auto"><li class="nav-item dropdown"><a aria-expanded="false" aria-haspopup="true" class="nav-link dropdown-toggle" data-toggle="dropdown" href="#" id="navbarProject InspirationDropdown" role="button">Project Inspiration</a><div aria-labelledby="navbarProject InspirationDropdown" class="dropdown-menu"><a class="dropdown-item" href="https://2021.igem.org/Team:SUNY_Oneonta/Inspiration">Inspiration</a><a class="dropdown-item" 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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><h2>RPA Figure 6 Re-do</h2><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><h2>Repeat Experiment from June 6th</h2><p>The following samples had their nanogram concentrations recorded in the table below.</p><div class="image"><img alt="Positive and Negative Controls at Different Temperatures" src="https://static.igem.org/mediawiki/2021/4/44/T--SUNY_Oneonta--img--RPALN-Fig6Redo_0608.png" style="width: 75%"/></div><p>*Gel 10µL of unclean RPA product 1kb + MWL measured up at where the product should be</p><hr/><h1>June 9th, 2021</h1><h2>RPA Reaction Cleaned up DNA Core</h2><p>Nanogram concentrations of samples were recorded</p><div class="image"><img alt=" " src="https://static.igem.org/mediawiki/2021/6/6f/T--SUNY_Oneonta--img--RPALN-NanogramConcentrations_0609.png" style="width: 75%"/></div><hr/><h1>June 10th, 2021</h1><h2>RPA Clean Trial 3</h2><p>Nanogram concentrations of samples were recorded</p><div class="image"><img alt=" " src="https://static.igem.org/mediawiki/2021/6/62/T--SUNY_Oneonta--img--RPALN-NanogramConcentrations3_0610.png" style="width: 75%"/></div><hr/><h1>June 16th, 2021</h1><h2>Repeat Experiment from June 7th using a <strong>NEW</strong> Twist-DX Kit</h2><div class="image"><img alt=" " src="https://static.igem.org/mediawiki/2021/7/7e/T--SUNY_Oneonta--img--RPALN-Fig6Redo_0616.png" style="width: 75%"/></div><ul><li>The reaction will be set up using the protocol posted on the iGEM Teams Files.</li></ul><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><li>Samples were incubated for 40 minutes at their respective temperatures.</li></ul><p><strong>PCR Cleanup:</strong></p><ul><li>45µL RPA Product<ul><li>40µL elution buffer</li></ul></li></ul><p><strong>Gel:</strong> 0.4g agarose 50mL 1x TBE</p><ul><li>1kb ladder</li><li>2F,10R samples: <strong>DILUTED:</strong> 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye</li><li>4F,11R samples: <strong>DILUTED:</strong> 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye</li><li>10µL of each sample mixture to each well</li></ul><p><strong>To Do:</strong></p><ul><li>Repeat gel using clean RPA product, do NOT dilute.</li></ul><hr/><h1>June 23rd, 2021</h1><h2>Repeat Experiment from June 7th using a <strong>NEW</strong> Twist-DX Kit</h2><p>New dNTP Stock Solution to be prepared...</p><table><thead><tr><th style="text-align:center">Reagent</th><th style="text-align:center">Stock</th><th style="text-align:center">Working</th></tr></thead><tbody><tr><td style="text-align:center">dNTPs</td><td style="text-align:center">100mM</td><td style="text-align:center">10mM</td></tr></tbody></table><p>$$(100mM)(v_{1}) = (10mM)(100µL)$$ $$v_{1} = 10µL$$</p><ul><li><p>10µL of each dNTP stock will be added, in addition to 60µL of nuclease-free water to bring the final volume to 100µL</p></li><li><p>New working primers will be prepared.</p></li><li><p>diluted primers</p><ul><li>10_RPA_R</li><li>11_RPA_R</li><li>4_RPA_F</li><li>2_RPA_F</li></ul></li><li><p>100mM -> all diluted with 10µL of primer + 90µL of nuclease-free (NF) water</p></li></ul><p>The following set up will be used:</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> | | </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><h2>RPA Figure 6 Re-do</h2><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><h2>Repeat Experiment from June 6th</h2><p>The following samples had their nanogram concentrations recorded in the table below.</p><div class="image"><img alt="Positive and Negative Controls at Different Temperatures" src="https://static.igem.org/mediawiki/2021/4/44/T--SUNY_Oneonta--img--RPALN-Fig6Redo_0608.png" style="width: 75%"/></div><p>*Gel 10µL of unclean RPA product 1kb + MWL measured up at where the product should be</p><hr/><h1>June 9th, 2021</h1><h2>RPA Reaction Cleaned up DNA Core</h2><p>Nanogram concentrations of samples were recorded</p><div class="image"><img alt=" " src="https://static.igem.org/mediawiki/2021/6/6f/T--SUNY_Oneonta--img--RPALN-NanogramConcentrations_0609.png" style="width: 75%"/></div><hr/><h1>June 10th, 2021</h1><h2>RPA Clean Trial 3</h2><p>Nanogram concentrations of samples were recorded</p><div class="image"><img alt=" " src="https://static.igem.org/mediawiki/2021/6/62/T--SUNY_Oneonta--img--RPALN-NanogramConcentrations3_0610.png" style="width: 75%"/></div><hr/><h1>June 16th, 2021</h1><h2>Repeat Experiment from June 7th using a <strong>NEW</strong> Twist-DX Kit</h2><div class="image"><img alt=" " src="https://static.igem.org/mediawiki/2021/7/7e/T--SUNY_Oneonta--img--RPALN-Fig6Redo_0616.png" style="width: 75%"/></div><ul><li>The reaction will be set up using the protocol posted on the iGEM Teams Files.</li></ul><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><li>Samples were incubated for 40 minutes at their respective temperatures.</li></ul><p><strong>PCR Cleanup:</strong></p><ul><li>45µL RPA Product<ul><li>40µL elution buffer</li></ul></li></ul><p><strong>Gel:</strong> 0.4g agarose 50mL 1x TBE</p><ul><li>1kb ladder</li><li>2F,10R samples: <strong>DILUTED:</strong> 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye</li><li>4F,11R samples: <strong>DILUTED:</strong> 2µL eluted product, 8µL Nuclease-free water, 2µL of 6x Loading Dye</li><li>10µL of each sample mixture to each well</li></ul><p><strong>To Do:</strong></p><ul><li>Repeat gel using clean RPA product, do NOT dilute.</li></ul><hr/><h1>June 23rd, 2021</h1><h2>Repeat Experiment from June 7th using a <strong>NEW</strong> Twist-DX Kit</h2><p>New dNTP Stock Solution to be prepared...</p><table><thead><tr><th style="text-align:center">Reagent</th><th style="text-align:center">Stock</th><th style="text-align:center">Working</th></tr></thead><tbody><tr><td style="text-align:center">dNTPs</td><td style="text-align:center">100mM</td><td style="text-align:center">10mM</td></tr></tbody></table><p>$$(100mM)(v_{1}) = (10mM)(100µL)$$ $$v_{1} = 10µL$$</p><ul><li><p>10µL of each dNTP stock will be added, in addition to 60µL of nuclease-free water to bring the final volume to 100µL</p></li><li><p>New working primers will be prepared.</p></li><li><p>diluted primers</p><ul><li>10_RPA_R</li><li>11_RPA_R</li><li>4_RPA_F</li><li>2_RPA_F</li></ul></li><li><p>100mM -> all diluted with 10µL of primer + 90µL of nuclease-free (NF) water</p></li></ul><p>The following set up will be used:</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> |