Difference between revisions of "Team:CCU Taiwan/Notebook"

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<div class="backgroundNotebook">
 
<div class="backgroundNotebook">
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<div class="photoNotebook"><h1 class="bigtitle">NOTEBOOK<h1></div>
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<div class="photoExperiments"><h1 class="bigtitle">EXPERIMENTS<h1></div>
 
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       <div class="content">
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<iframe src=”http://docs.google.com/gview?url=https://drive.google.com/file/d/1QRCVtDL3mHJBKcHj6BLPGNw94op-5GhW/view?usp=sharing&embedded=true” style=”width:600px;” frameborder=”0″></iframe>
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<br><br>
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<p class="second">February</p>
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<p class="description">Molecular biology class<br>E-coli genomic DNA preparation<br>E-coli transformation</p>
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<p class="second">March</p>
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<p class="description">Instrument operation</p>
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<p class="second">April</p>
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<p class="description">Project brainstorming-Product Positioning, HGS monomer proportion</p>
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<p class="second">May</p>
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<p class="description">Culture selection-compare Yeast, E-coli, Acetobacter aceti<br>Gene design</p>
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<p class="second">June</p>
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<p class="description">Interlab experiment-Calbration1,2,3</p>
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<p class="second">July</p>
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<p class="description">7/3<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Start interlab experiment-cell measurement<br>7/5<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;YPD formulation<br>7/9<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Yeast(X33) culture<br>7/13<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fundraising briefing session<br>7/16<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Communicate with NCKU(interlab &
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project)<br>7/18<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Communicate with BIT(project)</p>
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<p class="description">8/5<br>19:00-20:00<br>&nbsp;&nbsp;Day 2:<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pick 2 colonies from each of the transformation plates and inoculate in 5mL LB medium +Chloramphenicol. Grow the cells overnight (14 hours) at 37°C and 220 rpm.</p>
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<p class="description">8/6<br>10:00-18:00<br>&nbsp;&nbsp;Day 3: Cell growth, sampling, and assay<br>
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<ol>
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<li>Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)</li>
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<li>Measure Abs600 of these 1:10 diluted cultures</li>
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<li>Record the data in your notebook<img src="https://static.igem.org/mediawiki/2018/f/f1/T--CCU_Taiwan--8.6.1.png"></img></li>
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<li>Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).<img src="https://static.igem.org/mediawiki/2018/f/f7/T--CCU_Taiwan--8.6.2.png"></img></li>
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<li>Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.</li>
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<li>Measure your samples (Abs600 and Fluorescence measurement)<img src="https://static.igem.org/mediawiki/2018/f/f1/T--CCU_Taiwan--8.6.1.png"></img></li>
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</ol>
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</p>
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<br><br>
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<p class="description">Digest pGAPZ A(X3)/ pUCIDT_Lac1/ pUCIDT_Px16/ pUCIDT_Px18 with AgeI,EcoRI</P>
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<br><br>
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<p class="description">8/7<br>10:00-12:00<br>Fluorescence measurement</p>
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<p class="description">Ligation</p>
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<p class="description">8/8<br>&nbsp;&nbsp;Day 2:<br> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pick 2 colonies from each of the transformation plates and inoculate in 5mL LB medium +Chloramphenicol. Grow the cells overnight (16 hours) at 37°C and 220 rpm.<br>Transformation
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<ol>
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<li>Add all pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18 (15µl) into 20µl ECOS™ 101 Competent Cells [DH5a]</li>
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<li>Incubate on ice 5 minutes</li>
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<li>Heat shock at 42℃ 45 second</li>
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<li>Incubate on ice 5 minutes</li>
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<li>Add 140µl LB</li>
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<li>Incubate at 37℃ 1hr</li>
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<li>Spread on LB+ Zeocin plate</li>
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</ol>
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</p>
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<p class="description">Ligation</p>
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<p class="description">8/9<br>10:00-16:00<br>&nbsp;&nbsp;Day 3:Cell growth, sampling, and assay
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<ol>
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<li>Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)</li>
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<li>Measure Abs600 of these 1:10 diluted cultures</li>
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<li>Record the data in your notebook</li>
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<li>Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).</li>
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<li>Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.</li>
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<li>Measure your samples (Abs600 and Fluorescence measurement)</li>
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<li>Spread on LB+ Zeocin plate</li>
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</ol>
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</p>
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<p class="description">Transformation <br>
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<ol>
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<li>Add all pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18 (20µl) into 20µl Competent Cells DH5a and pGAPZ A 1µl+19µl ddH2O into 10µl Competent Cells DH5a as positive control.</li>
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<li>Incubate on ice 15min</li>
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<li>Heat shock at 42℃ 45sec</li>
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<li>Incubate on ice 5min</li>
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<li>Add 140µl LB</li>
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<li>Incubate at 37℃ 1hr</li>
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<li>Spread on LB+ Zeocin plate</li>
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</ol>
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</p>
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<p class="description">8/10<br>10:00-16:00<br>&nbsp;&nbsp;Digest pUCIDT_Lac1/ pUCIDT_Px16/ pUCIDT_Px18 with AgeI,EcoRI</p>
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<p class="description">Gel Extraction</p>
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<p class="description">8/11<br>Ligation</p>
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<br><br>
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<p class="description">8/12<br>Transformation<br>pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18</p>
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<p class="description">8/13<br>pGAPZ-A_Lac1-pGAPZ-A_Px16-pGAPZ-A_Px18-clone<br>20180813 PCR (pGAPZ-A_Lac1, pGAPZ-A_Px16, pGAPZ-A_Px18)</p>
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<p class="description">8/14<br>Miniprep<br>Result</p>
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<p class="description">8/15<br>12:00 Digest (use AgeI, EcoRI)<br>Protocol reference:  </p><a href="https://nebcloner.neb.com/#!/protocol/re/sequential-heat/AgeI,EcoRI">https://nebcloner.neb.com/#!/protocol/re/sequential-heat/AgeI,EcoRI</a>
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<p class="description">8/22</p>
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<p class="description">8/23</p>
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<p class="description">8/24</p>
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<p class="description">8/27</p>
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<p class="description">8/28<br>Transform
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<ol>
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<li>pGAPZA-1 + His4 + Px16</li>
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<li>pGAPZA-1 + His4 + Px18</li>
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<li>pGAPZA-1 + His4 + Lac1<br>LB + zeocin</li>
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</ol>
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</p>
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<br><br>
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<p class="description">8/29</p>
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<p class="description">8/30<br>Check<br>pGAPZA-1 + His4<br>1. Digest - AgeI</p>
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<br><br><br>
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Revision as of 08:17, 16 October 2018

EXPERIMENTS