Difference between revisions of "Team:ECNUAS/Notebook"

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    <title>ECNUAS</title>
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<h1>Notebook</h1>
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</head>
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<p> Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.</p>
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</div>
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<body>
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<div class="clear"></div>
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    <nav class="head-nav clearfix">
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        <div class="top-block"></div>
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        <div class="top-nav-bar">
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            <ul class="clearfix">
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<div class="column two_thirds_size">
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                <span class="small-logo"></span>
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<h3>What should this page have?</h3>
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                <li>
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<ul>
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                    <a href="https://2021.igem.org/Team:ECNUAS">Home</a>
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<li>Chronological notes of what your team is doing.</li>
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                </li>
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<li> Brief descriptions of daily important events.</li>
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                <li class="active">
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<li>Pictures of your progress. </li>
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                    <a href="">Project</a>
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<li>Mention who participated in what task.</li>
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                    <div class="sub-nav">
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</ul>
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                        <ul>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Description"
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</div>
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                                    class="sub-nav-74">Description</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Experiments"
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<div class="column third_size">
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                                    class="sub-nav-74">Experiments</a></li>
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<div class="highlight decoration_A_full">
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Results" class="sub-nav-74">Results</a></li>
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<h3>Inspiration</h3>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Proof_Of_Concept" class="sub-nav-52">Proof Of
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<p>You can see what others teams have done to organize their notes:</p>
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                                    Concept</a></li>
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                            <li class="current-sub-nav"><a href="#" class="sub-nav-52">Notebook</a>
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<ul>  
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                            </li>
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<li><a href="https://2018.igem.org/Team:Munich/Notebook">2018 Munich</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Safety">Safety</a></li>
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                        </ul>
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<li><a href="https://2019.igem.org/Team:Georgia_State/Notebook">2019 Georgia State</a></li>
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                    </div>
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<li><a href="https://2019.igem.org/Team:Newcastle/Notebook">2019 Newcastle</a></li>
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                </li>
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                <li>
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                    <a href="">Parts</a>
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<li><a href="https://2020.igem.org/Team:IISER-Pune-India/Notebook">2020 IISER Pune India</a></li>
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                    <div class="sub-nav">
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<li><a href="https://2020.igem.org/Team:Lund/Notebook">2020 Lund</a></li>
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                        <ul>
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<li><a href="https://2020.igem.org/Team:NOVA_LxPortugal/Notebook">2020 NOVA LxPortugal</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Collection" class="sub-nav-74">Parts
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<li><a href="https://2020.igem.org/Team:RDFZ-China/NoteBook">2020 RDFZ China</a></li>
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                                    Collection</a></li>
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</ul>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Engineering"
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</div>
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                                    class="sub-nav-74">Engineering</a></li>
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</div>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Contribution"
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                                    class="sub-nav-74">Contribution</a></li>
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                        </ul>
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                    </div>
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                </li>
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                <li>
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                    <a href="">Human Practices</a>
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                    <div class="sub-nav">
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                        <ul>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Human_Practices"
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                                    class="sub-nav-74">Integrated Human Practice</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Communication"
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                                    class="sub-nav-74">Communication</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Fundraising"
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                                    class="sub-nav-74">Fundraising</a></li>
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                        </ul>
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                    </div>
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                </li>
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                <li>
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                    <a href="https://2021.igem.org/Team:ECNUAS/Implementation">Implementation</a>
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                </li>
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                <li>
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                    <a href="https://2021.igem.org/Team:ECNUAS/Entrepreneurship">Entrepreneurship</a>
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                </li>
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                <li>
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                    <a href="https://2021.igem.org/Team:ECNUAS/Model">Model</a>
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                </li>
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                <li>
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                    <a href="">Team</a>
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                    <div class="sub-nav">
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                        <ul>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Members" class="sub-nav-74">Team
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                                    Members</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Attributions"
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                                    class="sub-nav-74">Attributions</a></li>
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                            <li><a href="https://2021.igem.org/Team:ECNUAS/Collaborations"
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                                    class="sub-nav-74">Collaborations</a></li>
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                        </ul>
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                    </div>
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                </li>
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            </ul>
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        </div>
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    </nav>
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    <div class="sub-banner">
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        <img src="https://static.igem.org/mediawiki/2021/e/ed/T--ECNUAS--parts_collection02.png" alt="" />
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    </div>
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    <div class="sub-content">
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        <div class="sub-title">Notebook</div>
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        <div class="article-title">2021-7-15</div>
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        <div class="article-content">- Ice-breaking</div>
 +
        <div class="article-content">- Initial Discussion on the project plan</div>
 +
        <div class="img-wrap no-margin">
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            <img src="https://static.igem.org/mediawiki/2021/e/e7/T--ECNUAS--notebook01.jpg" alt="">
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        </div>
 +
        <div class="article-title">2021-7-16</div>
 +
        <div class="article-content">- Subject background survey</div>
 +
        <div class="article-content">- Basic experimental operation</div>
 +
        <div class="article-content">- Laboratory safety training</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/0/0a/T--ECNUAS--notebook02.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- Prepare growing medium (liquid culture medium)</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/e/e6/T--ECNUAS--notebook03.jpg" alt="">
 +
        </div>
 +
        <div class="article-title">2021-7-17 </div>
 +
        <div class="article-content">- Basic operate training </div>
 +
        <div class="article-content">- Learning the concept of recombinant plasmids</div>
 +
        <div class="article-content">- Prepare growing medium (solid culture medium)</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            • One type of Kanamycin
 +
            • One type of Ampicillin
 +
            • One type of Kanamycin and Ampicillin
 +
        </div>
 +
        <div class="article-content">- Dilute TAE buffer</div>
 +
        <div class="article-title">2021-7-18</div>
 +
        <div class="article-content">- Restriction endonuclease reaction</div>
 +
        <div class="article-content">- DNA agarose gel electrophoresis identification</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/c/c2/T--ECNUAS--notebook04.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- DNA agarose gel reavers and purification</div>
 +
        <div class="article-content">- DNA clip connection reaction</div>
 +
        <div class="article-content">- Plasmid transformation</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            • Plasmid A (Atz sensor plasmid):pET28a(+)_P7_AtzR
 +
            • Plasmid B (Atz reporter plasmid): pUC57-pro_Pprovoin5_amilGFP
 +
        </div>
 +
        <div class="article-title">2021-7-19</div>
 +
        <div class="article-content">- Found the plasmid B’s plate didn’t grow any colony, but Plasmid A grew</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/2/2f/T--ECNUAS--notebook05.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- Amplification of Atz sensor plasmid and leave it in shaker through night</div>
 +
        <div class="article-content">- Then we start to think why plasmid B didn’t grow any flora</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            • We think that the plasmid transformation in day 7-18 might got some problem, so we do the DNA clip
 +
            connection and Plasmid transformation again by using day 7-18 DNA clip connection reaction.
 +
        </div>
 +
        <div class="article-content">- So we repeat experience again (but we didn’t get good stripes)</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            • restriction endonuclease reaction (only Pprovoin5)
 +
        </div>
 +
        <div class="article-content" style="padding-left: 60px; box-sizing: border-box;">
 +
            - Increase the amount of sample to 15 μL<br />
 +
            - Increase the amount of Pprovoin5 enzyme to 1.25 μL
 +
        </div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            • DNA agarose gel electrophoresis identification (all result get very light stripes)
 +
        </div>
 +
        <div class="article-content" style="padding-left: 60px; box-sizing: border-box;">
 +
            1. First gel is from day 7-18 DNA clip connection reaction
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/f/ff/T--ECNUAS--notebook06.jpg" alt="">
 +
        </div>
 +
        <div class="article-content" style="padding-left: 60px; box-sizing: border-box;">
 +
            2. Second gel is from day 7-19 restriction endonuclease digestion product
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/6/63/T--ECNUAS--notebook07.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- The potential cause why we don’t get stripes or very light stripes</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            A. turnip the gel 180 (may cause the stripe light)<br />
 +
            B. When we’re adding more TAE, the TAE added might disperse light<br />
 +
            C. Adding too much sample might disperse the stripes (because we change from 10 μL to 15 μL
 +
        </div>
 +
        <div class="article-content">- Preparing LB medium</div>
 +
        <div class="article-title">2021-7-20</div>
 +
        <div class="article-content">- Continue plasmid extraction</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">• By using the colonies from
 +
            day 7-19 (plasmid A)</div>
 +
        <div class="article-content">- Restriction endonuclease reaction and DNA agarose gel electrophoresis
 +
            identification (only Pprovoin5) (twice because first one has no stripe)</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">1. No change</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/4/4d/T--ECNUAS--notebook08.jpg" alt="">
 +
        </div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">2. Changing the system from
 +
            50μL to 40μL by decrease water (10μL)</div>
 +
        <div class="article-content">- DNA agarose gel electrophoresis identification result</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            1. No/very light stripe<br />
 +
            2. Stripe appeared
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/5/5e/T--ECNUAS--notebook09.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- DNA clip connection reaction</div>
 +
        <div class="article-content">- Plasmid transformation</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            A. Restriction endonuclease reaction product following the protocol (no resistance)<br />
 +
            B. Restriction endonuclease reaction product following the protocol (with resistance)<br />
 +
            C. 40 μL system restriction endonuclease reaction product<br />
 +
            D. 40 μL system restriction endonuclease reaction product<br />
 +
            E. Plasmid transformation from day 7-18
 +
        </div>
 +
        <div class="article-title">2021-7-21</div>
 +
        <div class="article-content">- No conoly from day 7-20 (ABCDE)</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/a/ac/T--ECNUAS--notebook10.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- DNA agarose gel electrophoresis identification of day 7-20 DNA connection
 +
            reaction, found that there’s no stripes (very light)</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/d/d6/T--ECNUAS--notebook11.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- Clip connection</div>
 +
        <div class="article-content">- Plasmid transformation (change from 10min RTP to 20 Celsius 1h)</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/6/66/T--ECNUAS--notebook12.jpg" alt="">
 +
        </div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">
 +
            F. E. coli without transfer in plasmid in the non-resistant LB medium to make sure E. Coli sill
 +
            working<br />
 +
            G. Transfer in Pprovoin5 plasmid<br />
 +
            H. Plasmid transformation from 7-21<br />
 +
            I. Plasmid transformation from 7-21<br />
 +
            J. Plasmid transformation from 7-21<br />
 +
        </div>
 +
        <div class="article-title">2021-7-22</div>
 +
        <div class="article-content">- No conoly until afternoon second one from 7-21 has some colony, so plasmid
 +
            extraction 16h amplification</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/f/f4/T--ECNUAS--notebook13.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- Restriction endonuclease reaction</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">• Leave it in 37 celsius
 +
            through night</div>
 +
        <div class="article-content">- DNA clip connection reaction (increase the amount of plasmid and leave 16 Celsius
 +
            through night)</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">• AmiGFP: Pprovoin5 = 4.5 μL :
 +
            1.5 μL</div>
 +
        <div class="article-title">2021-7-23</div>
 +
        <div class="article-content">- Plasmid extraction from day 7-22 amplification</div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">• So we could have more
 +
            Pprovoin5 plasmid</div>
 +
        <div class="article-content">- DNA agarose gel electrophoresis identification </div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">• Marker changed</div>
 +
        <div class="article-content" style="padding-left: 60px; box-sizing: border-box;">
 +
            - 5 μL marker<br />
 +
            - 0 μL loading buffer<br />
 +
            - 2 μL Nucleic acid dyestuff
 +
        </div>
 +
        <div class="article-content" style="padding-left: 30px; box-sizing: border-box;">• Negative control (only amiGFP
 +
            plasmid because we don’t have enough Pprovoin5 plasmid)</div>
 +
        <div class="article-title">2021-9</div>
 +
        <div class="article-content">
 +
            - Repeat the plasmid B constrcution<br />
 +
            - Coloy PCR to verify the transformants of plasmid B and extract the plasmid for sequencing<br />
 +
            - Prepare LB medium with two antibiotics (kana&amp)<br />
 +
            - Extract the plasmid A and plasmid B to be transformed into DH5a to obtain bacteria C
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/3/3e/T--ECNUAS--notebook14.jpg" alt="">
 +
        </div>
 +
        <div class="article-title">2021-10-1~7</div>
 +
        <div class="article-content">- Conduct function tests</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/1/1f/T--ECNUAS--notebook15.jpg" alt="">
 +
        </div>
 +
        <div class="article-content">- Cell-free extraction experiments</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/d/d4/T--ECNUAS--notebook16.jpg" alt="">
 +
        </div>
 +
    </div>
 +
    <footer class="footer">
 +
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Revision as of 18:31, 16 October 2021

ECNUAS

Notebook
2021-7-15
- Ice-breaking
- Initial Discussion on the project plan
2021-7-16
- Subject background survey
- Basic experimental operation
- Laboratory safety training
- Prepare growing medium (liquid culture medium)
2021-7-17
- Basic operate training
- Learning the concept of recombinant plasmids
- Prepare growing medium (solid culture medium)
• One type of Kanamycin • One type of Ampicillin • One type of Kanamycin and Ampicillin
- Dilute TAE buffer
2021-7-18
- Restriction endonuclease reaction
- DNA agarose gel electrophoresis identification
- DNA agarose gel reavers and purification
- DNA clip connection reaction
- Plasmid transformation
• Plasmid A (Atz sensor plasmid):pET28a(+)_P7_AtzR • Plasmid B (Atz reporter plasmid): pUC57-pro_Pprovoin5_amilGFP
2021-7-19
- Found the plasmid B’s plate didn’t grow any colony, but Plasmid A grew
- Amplification of Atz sensor plasmid and leave it in shaker through night
- Then we start to think why plasmid B didn’t grow any flora
• We think that the plasmid transformation in day 7-18 might got some problem, so we do the DNA clip connection and Plasmid transformation again by using day 7-18 DNA clip connection reaction.
- So we repeat experience again (but we didn’t get good stripes)
• restriction endonuclease reaction (only Pprovoin5)
- Increase the amount of sample to 15 μL
- Increase the amount of Pprovoin5 enzyme to 1.25 μL
• DNA agarose gel electrophoresis identification (all result get very light stripes)
1. First gel is from day 7-18 DNA clip connection reaction
2. Second gel is from day 7-19 restriction endonuclease digestion product
- The potential cause why we don’t get stripes or very light stripes
A. turnip the gel 180 (may cause the stripe light)
B. When we’re adding more TAE, the TAE added might disperse light
C. Adding too much sample might disperse the stripes (because we change from 10 μL to 15 μL
- Preparing LB medium
2021-7-20
- Continue plasmid extraction
• By using the colonies from day 7-19 (plasmid A)
- Restriction endonuclease reaction and DNA agarose gel electrophoresis identification (only Pprovoin5) (twice because first one has no stripe)
1. No change
2. Changing the system from 50μL to 40μL by decrease water (10μL)
- DNA agarose gel electrophoresis identification result
1. No/very light stripe
2. Stripe appeared
- DNA clip connection reaction
- Plasmid transformation
A. Restriction endonuclease reaction product following the protocol (no resistance)
B. Restriction endonuclease reaction product following the protocol (with resistance)
C. 40 μL system restriction endonuclease reaction product
D. 40 μL system restriction endonuclease reaction product
E. Plasmid transformation from day 7-18
2021-7-21
- No conoly from day 7-20 (ABCDE)
- DNA agarose gel electrophoresis identification of day 7-20 DNA connection reaction, found that there’s no stripes (very light)
- Clip connection
- Plasmid transformation (change from 10min RTP to 20 Celsius 1h)
F. E. coli without transfer in plasmid in the non-resistant LB medium to make sure E. Coli sill working
G. Transfer in Pprovoin5 plasmid
H. Plasmid transformation from 7-21
I. Plasmid transformation from 7-21
J. Plasmid transformation from 7-21
2021-7-22
- No conoly until afternoon second one from 7-21 has some colony, so plasmid extraction 16h amplification
- Restriction endonuclease reaction
• Leave it in 37 celsius through night
- DNA clip connection reaction (increase the amount of plasmid and leave 16 Celsius through night)
• AmiGFP: Pprovoin5 = 4.5 μL : 1.5 μL
2021-7-23
- Plasmid extraction from day 7-22 amplification
• So we could have more Pprovoin5 plasmid
- DNA agarose gel electrophoresis identification
• Marker changed
- 5 μL marker
- 0 μL loading buffer
- 2 μL Nucleic acid dyestuff
• Negative control (only amiGFP plasmid because we don’t have enough Pprovoin5 plasmid)
2021-9
- Repeat the plasmid B constrcution
- Coloy PCR to verify the transformants of plasmid B and extract the plasmid for sequencing
- Prepare LB medium with two antibiotics (kana&amp)
- Extract the plasmid A and plasmid B to be transformed into DH5a to obtain bacteria C
2021-10-1~7
- Conduct function tests
- Cell-free extraction experiments
Contact Info

WWeChat Official Account: Silent Spring

Email Contact: samlishensheng@qq.com