Team:Kyoto/Notebook

From 2011.igem.org

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(Week7: Monday 12th September - Sunday 18th September)
 
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{{Kyoto_Foreground}}
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{{Kyoto_Foreground|active_page=notebook}}
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{{Kyoto_Background}}
{{Kyoto_WikiDesign}}
{{Kyoto_WikiDesign}}
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<!-- #main -->
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= Notebook =
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<div id="main">
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= Lab Note =
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See our daily progress with the project !
<html>
<html>
<style>
<style>
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#index {
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a.note_link:visited {
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    margin:10px 0;  
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  color: #ffffff;
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    padding:0; 
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}
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    width:700px; 
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a.note_link {
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    height:30px; 
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    overflow:hidden; 
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#index li { list-style:none; width:68px; float:left; } 
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#index li a { 
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    display:block; 
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    line-height:30px; 
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    text-align:center; 
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}
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#index li a {
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  display: block;
  display: block;
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  line-height:30px;
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margin: 10px;
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width: 375px;
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height: 50px;
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  line-height:50px;
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text-align: center;
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background-color: #0000ff;
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color: #ffffff;
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font-size: 15px;
}
}
</style>
</style>
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<ul id="index">
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<li><a href="#lab-week1">Week1</a></li>
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<a href="https://2011.igem.org/Team:Kyoto/Lab Work" class="note_link">Lab Work</span></a>
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<li><a href="#lab-week2">Week2</a></li>
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<a href="https://2011.igem.org/Team:Kyoto/Diary" class="note_link">Diary</span></a>
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<li><a href="#lab-week3">Week3</a></li>
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<li><a href="#lab-week4">Week4</a></li>
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<li><a href="#lab-week5">Week5</a></li>
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<li><a href="#lab-week6">Week6</a></li>
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<li><a href="#lab-week7">Week7</a></li>
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<li><a href="#lab-week8">Week8</a></li>
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<li><a href="#lab-week9">Week9</a></li>
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<li><a href="#protocol">Protocol</a></li>
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</ul>
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</html>
</html>
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<html><a name="lab-week1"></a></html>
 
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== Week1: Monday 1st - Sunday 7th August ==
 
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'''Monday'''<br/>
 
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行った実験名:
 
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使った試薬名、容量:
 
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用いた機械:
 
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行った人:
 
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week2"></a></html>
 
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== Week2: Monday 8th - Sunday 14th August ==
 
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week3"></a></html>
 
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== Week3: Monday 15th - Sunday 21th August ==
 
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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Nutritional Signal(Sugiura,Shimosaka & Okumura)<br/>
 
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PCR Amplification of glnL and glnG from gDNA of E.coli<br/>
 
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--Primers--<br/>
 
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glnL<br/>
 
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Left primer: tctagaggagactgctttatggcaac<br/>
 
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Right primer: actagtaggaactatcgtcatcgactac<br/>
 
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glnG<br/>
 
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Left primer: tctagaggtgacgtttatgcaacga<br/>
 
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Right primer: actagtacacacaagctgtgaatcactc<br/>
 
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annealing temperature was 55 degrees.<br/>
 
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[[File:Kyoto-Gel0818.jpg]]<br/>
 
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lane 1,2 &7,8: DNA ladder(λDNA digested Hindlll,100bp), lane 3,4: glnG1,glnG2, lane 5,6: glnL1,glnL2<br/>
 
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After purification, the concentration of DNA are<br/>
 
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glnG1: 127.8ng/ul<br/>
 
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glnG2: 118.1ng/ul<br/>
 
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glnL1: 137.4ng/ul<br/>
 
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glnL2: 124.2ng/ul<br/>
 
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'''Friday'''<br/>
 
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Nutritional Signal(Shimosaka)<br/>
 
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Restriction of glnG1 and glnG2<br/>
 
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Cut them with Xbal and Spel for 2 hours at 37 degrees.<br/>
 
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Then, gel extraction of digested.<br/>
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week4"></a></html>
 
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== Week4: Monday 22th - Sunday 28th August ==
 
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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Luminescence(Kusaba, Terada, Hara):ハエの走行性実験① ♂、紫外線×2回、緑×2回 ♀、紫外線×2回、緑×2回
 
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'''Friday'''<br/>
 
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Luminescence(Kusaba):ハエの走行性実験① ♂、赤外線×2回 ♀、赤外線×2回
 
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Nutritional Signal(Hashiya):
 
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Transformation of bellow parts.<br/>
 
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4-17M:BBa_K325909(lux operon)<br/>
 
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1-12M:BBa_E0240<br/>
 
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2-17F:BBa_120260(low copy vector)<br/>
 
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PCR amplification of
 
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'''Saturday'''<br/>
 
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Luminescence(Kusaba):ハエの走行性実験① ♂、赤×2回、青×2回 ♀、赤×2回、青×2回
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week5"></a></html>
 
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== Week5: Monday 29th August - Sunday 4th September ==
 
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・PCR amplification of glnL and glnG from PCR products,glnL1 and glnG1.<br/>
 
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 --Primers--<br/>
 
-
 glnL<br/>
 
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 Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac<br/>
 
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 Right primer: ggactagtaggaactatcgtcatcgactac<br/>
 
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 glnG<br/>
 
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 Left primer: ggaattcgcggccgcttctagaggtgacgtttatgcaacga<br/>
 
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 Right primer: ggactagtacacacaagctgtgaatcactc<br/>
 
-
 annealing temperature was 55 degrees.<br/>
 
-
 
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・PCR amplification of glnL+G and rpoN from gDNA of E.coli.<br/>
 
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 --Primers--<br/>
 
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 glnL+G<br/>
 
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 Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac<br/>
 
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 Right primer: ggactagtacacacaagctgtgaatcactc<br/>
 
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 rpoN<br/>
 
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 Left primer: ggaattcgcggccgcttctagaggttctgaacatgaagcaa<br/>
 
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 Right primer: ggactagtatccttatcggttgggtca<br/>
 
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 annealing temperature was 56 degrees.<br/>
 
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-
 [[File:Kyoto-Gel08300.jpg]]<br/>
 
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 lane1: 100bp DNA ladder, lane2:glnL, lane3:glnG, lane4:glnG+L, lane5:rpoN from gDNA, lane6:rpoN from ASKA clone<br/>
 
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 After purification, the concentration of DNA are<br/>
 
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 glnL: 122.3 ng/ul<br/>
 
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 glnG: 64.7 ng/ul<br/>
 
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 glnL+G: 106.7 ng/ul<br/>
 
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 rpoN from gDNA: 111.4 ng/ul<br/>
 
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'''Wednesday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・Screening PCR of σ54 promoter + pSB1A3<br/>
 
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 [[File:Kyoto-Gel08301.jpg]]<br/>
 
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 We cultured σ54 promoter5<br/>
 
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'''Friday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・Restriction of σ54 promoter5,glnL, glnG, glnL+G and rpoN<br/>
 
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 Cut them with EcoRl and Spel
 
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 After purification, the concentration of DNA were<br/>
 
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 σ54 promoter5: 23.6 ng/ul<br/>
 
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 glnL: 28.1 ng/ul<br/>
 
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 glnG: 26.3 ng/ul<br/>
 
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 glnL+G: 15.3 ng/ul<br/>
 
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 rpoN: 20.8 ng/ul<br/>
 
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・Ligation reaction<br/>
 
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 Ligated glnL, glnG and rpoN to pSB1K3.<br/>
 
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'''Thursday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・Screening PCR of glnL, glnG, glnL+G and rpoN<br/>
 
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glnL
 
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 [[File:Kyoto-Gel09020.jpg]]<br/>
 
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glnG
 
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 [[File:Kyoto-Gel09021.jpg]]<br/>
 
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glnL+G & rpoN
 
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 [[File:Kyoto-Gel09022.jpg]]<br/>
 
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 We cultured glnL5, glnG4<br/>
 
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'''Saturday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・Mini prep of glnL5 and glnG4<br/>
 
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 glnL5: 43.9 ng/ul<br/>
 
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 glnG4: 38.1 ng/ul<br/>
 
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・Screening PCR of rpoN
 
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 [[File:Kyoto-Gel09020.jpg]]<br/>
 
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Predation(Hashiya)<br/>
 
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・PCR amplification of glmS<br/>
 
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 --Primers--<br/>
 
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 left primer:ggaattcgcggccgcttctagagcaggttgaccgacaacgata<br/>
 
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 right primer:ggactagtacgcagggcatccatttat<br/>
 
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 [[File:Kyoto-Gel09030.jpg]]<br/>
 
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 lane1: 100bp DNA ladder, lane2: glmS from gDNA, lane3: glmS from ASKA clone<br/>
 
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・TA cloning of glmS<br/>
 
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 Ligated glmS from gDNA to pTA vector.<br/>
 
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'''Sunday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・Screening PCR of rpoN<br/>
 
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 [[File:Kyoto-Gel09020.jpg]]<br/>
 
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・Transformation of bellow parts<br/>
 
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 1-23L: BBa_B0015  (double terminator)<br/>
 
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 1-18E: BBa_J23101 (constitutive promoter)<br/>
 
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 1-18C: BBa_J23100 (constitutive promoter)<br/>
 
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<html><a name="lab-week6"></a></html>
 
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== Week6: Monday 5th September - Sunday 11th September ==
 
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'''Monday'''<br/>
 
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Nutritional Signal(Hashiya)<br/>
 
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・Screening PCR of glnL+G+double terminator<br/>
 
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'''Tuesday'''<br/>
 
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Luminescence(Kusaba, Hara):ハエの走行性実験②(②は改良版) ♂、緑×2回 ♀、青×1回
 
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'''Wednesday'''<br/>
 
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Luminescence(Hara):ハエの走行性実験② ♂、青×2回 ♀、緑×2回、青×1回
 
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'''Thursday'''<br/>
 
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Luminescence(Hara):ハエの走行性実験② ♂、紫外線×3回 ♀、紫外線×3回
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
 
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Luminescence(Kusaba, Hara):ハエの走行性実験② ♂、青×2回、赤×2回 ♀、青×2回、赤×2回 
 
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'''Sunday'''<br/>
 
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Luminescence(Kusaba, Hara):ハエの走行性実験② ♂、紫外線×2回、
 
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<html><a name="lab-week7"></a></html>
 
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== Week7: Monday 12th September - Sunday 18th September ==
 
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'''Monday'''<br/>
 
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Luminescence:大腸菌の形質転換(Hashiya) ハエの走行性実験②(Kusaba, Hara)
 
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'''Tuesday'''<br/>
 
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Luminescence:大腸菌はじめて光る。しかし光量は少ない。
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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Digestion(Kajita)
 
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PCR amplification of SAM-P20 and ChiA
 
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:We performed colony direct PCRs from a ''S. albogriseolus'' colony and a ''S. avermitilis'' colony.
 
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<table style="float:left;"><tr><td>
 
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:{|class="wikitable"
 
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|+Reaction mixture
 
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!width="100"|Component
 
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!width="100"|Volume(&mu;l)
 
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|-
 
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|2x Buffer||25
 
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|-
 
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|2mM dNTPs||10
 
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|-
 
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|Primer 1||1.5
 
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|-
 
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|Primer 2||1.5
 
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|-
 
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|Template||1
 
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|-
 
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|ddH<sub>2</sub>O||up to 50
 
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|}
 
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</td></tr></table>
 
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<table><tr><td>
 
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:{|class="wikitable"
 
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|+PCR condition
 
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|Predenature||94C||2m||
 
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|-
 
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|Denature||98C||10s||rowspan="3"|25cycles
 
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|-
 
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|Annealing||56C||30s
 
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|-
 
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|Extension||68C||1m30s
 
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|}
 
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</td></tr></table>
 
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<br style="clear: both;">
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week8"></a></html>
 
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== Week8: Monday 19th September - Sunday 25th September ==
 
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week9"></a></html>
 
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== Week9: Monday 26th September - Sunday 2nd October ==
 
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="protocol"></a></html>
 
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== Protocol ==
 
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=== Medium for drosophila ===
 
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::{| class="wikitable"
 
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|+ [http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]
 
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! width="300" |Materials
 
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! width="300" |Methods
 
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|-
 
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|
 
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* water : 500mL
 
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* dry yeast : 20g
 
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* corn flour : 45g
 
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* glucose : 50g
 
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* agarose : 3.5~5g
 
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* propionic acid : 1.5mL
 
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* 10% p-hydroxybenzoate in 70% Eternol : 5g
 
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|
 
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# Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it.
 
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# Stir corn flour and glucose with the remaining water.
 
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# Stir 1 and 2, then autoclave it again.
 
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# after autoclave, add propionic acid and 10% p-hydroxybenzoate in 70% Eternol into it.■
 
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|}
 
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</div>
 
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<!-- /#main -->
 

Latest revision as of 15:00, 27 September 2011

Notebook

See our daily progress with the project !

Lab Work Diary