|
|
(13 intermediate revisions not shown) |
Line 1: |
Line 1: |
- | {{Kyoto_Foreground}} | + | {{Kyoto_Foreground|active_page=notebook}} |
| + | {{Kyoto_Background}} |
| {{Kyoto_WikiDesign}} | | {{Kyoto_WikiDesign}} |
| | | |
- | <!-- #main -->
| + | = Notebook = |
- | <div id="main">
| + | |
| | | |
- | = Lab Note =
| + | See our daily progress with the project ! |
| | | |
| <html> | | <html> |
| <style> | | <style> |
- | #index {
| + | a.note_link:visited { |
- | margin:10px 0;
| + | color: #ffffff; |
- | padding:0;
| + | } |
- | width:700px;
| + | a.note_link { |
- | height:30px;
| + | |
- | overflow:hidden;
| + | |
- | }
| + | |
- | #index li { list-style:none; width:68px; float:left; } | + | |
- | #index li a {
| + | |
- | display:block;
| + | |
- | line-height:30px;
| + | |
- | text-align:center;
| + | |
- | } | + | |
- | | + | |
- | #index li a {
| + | |
| display: block; | | display: block; |
- | line-height:30px; | + | margin: 10px; |
| + | width: 375px; |
| + | height: 50px; |
| + | line-height:50px; |
| + | text-align: center; |
| + | background-color: #0000ff; |
| + | color: #ffffff; |
| + | font-size: 15px; |
| } | | } |
| </style> | | </style> |
- | <ul id="index">
| + | |
- | <li><a href="#lab-week1">Week1</a></li>
| + | <a href="https://2011.igem.org/Team:Kyoto/Lab Work" class="note_link">Lab Work</span></a> |
- | <li><a href="#lab-week2">Week2</a></li>
| + | <a href="https://2011.igem.org/Team:Kyoto/Diary" class="note_link">Diary</span></a> |
- | <li><a href="#lab-week3">Week3</a></li>
| + | |
- | <li><a href="#lab-week4">Week4</a></li>
| + | |
- | <li><a href="#lab-week5">Week5</a></li>
| + | |
- | <li><a href="#lab-week6">Week6</a></li>
| + | |
- | <li><a href="#lab-week7">Week7</a></li>
| + | |
- | <li><a href="#lab-week8">Week8</a></li>
| + | |
- | <li><a href="#lab-week9">Week9</a></li>
| + | |
- | <li><a href="#protocol">Protocol</a></li>
| + | |
- | </ul>
| + | |
| </html> | | </html> |
- |
| |
- | <html><a name="lab-week1"></a></html>
| |
- | == Week1: Monday 1st - Sunday 7th August ==
| |
- | '''Monday'''<br/>
| |
- | 行った実験名:
| |
- |
| |
- | 使った試薬名、容量:
| |
- |
| |
- | 用いた機械:
| |
- |
| |
- | 行った人:
| |
- |
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="lab-week2"></a></html>
| |
- |
| |
- | == Week2: Monday 8th - Sunday 14th August ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="lab-week3"></a></html>
| |
- | == Week3: Monday 15th - Sunday 21th August ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- | Nutritional Signal(Sugiura,Shimosaka & Okumura)<br/>
| |
- | PCR Amplification of glnL and glnG from gDNA of E.coli<br/>
| |
- |
| |
- | --Primers--<br/>
| |
- | glnL<br/>
| |
- | Left primer: tctagaggagactgctttatggcaac<br/>
| |
- | Right primer: actagtaggaactatcgtcatcgactac<br/>
| |
- | glnG<br/>
| |
- | Left primer: tctagaggtgacgtttatgcaacga<br/>
| |
- | Right primer: actagtacacacaagctgtgaatcactc<br/>
| |
- | annealing temperature was 55 degrees.<br/>
| |
- | [[File:Kyoto-Gel0818.jpg]]<br/>
| |
- | lane 1,2 &7,8: DNA ladder(λDNA digested Hindlll,100bp), lane 3,4: glnG1,glnG2, lane 5,6: glnL1,glnL2<br/>
| |
- |
| |
- | After purification, the concentration of DNA are<br/>
| |
- | glnG1: 127.8ng/ul<br/>
| |
- | glnG2: 118.1ng/ul<br/>
| |
- | glnL1: 137.4ng/ul<br/>
| |
- | glnL2: 124.2ng/ul<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- | Nutritional Signal(Shimosaka)<br/>
| |
- | Restriction of glnG1 and glnG2<br/>
| |
- | Cut them with Xbal and Spel for 2 hours at 37 degrees.<br/>
| |
- | Then, gel extraction of digested.<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="lab-week4"></a></html>
| |
- |
| |
- | == Week4: Monday 22th - Sunday 28th August ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- | 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
| |
- |
| |
- | '''Friday'''<br/>
| |
- | Luminescence(Kusaba):ハエの走行性実験① ♂、赤外線×2回 ♀、赤外線×2回
| |
- |
| |
- | Nutritional Signal(Hashiya):
| |
- | Transformation of bellow parts.<br/>
| |
- | 4-17M:BBa_K325909(lux operon)<br/>
| |
- | 1-12M:BBa_E0240<br/>
| |
- | 2-17F:BBa_120260(low copy vector)<br/>
| |
- |
| |
- | PCR amplification of
| |
- |
| |
- | '''Saturday'''<br/>
| |
- | Luminescence(Kusaba):ハエの走行性実験① ♂、赤×2回、青×2回 ♀、赤×2回、青×2回
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="lab-week5"></a></html>
| |
- |
| |
- | == Week5: Monday 29th August - Sunday 4th September ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・PCR amplification of glnL and glnG from PCR products,glnL1 and glnG1.<br/>
| |
- | --Primers--<br/>
| |
- | glnL<br/>
| |
- | Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac<br/>
| |
- | Right primer: ggactagtaggaactatcgtcatcgactac<br/>
| |
- | glnG<br/>
| |
- | Left primer: ggaattcgcggccgcttctagaggtgacgtttatgcaacga<br/>
| |
- | Right primer: ggactagtacacacaagctgtgaatcactc<br/>
| |
- | annealing temperature was 55 degrees.<br/>
| |
- |
| |
- | ・PCR amplification of glnL+G and rpoN from gDNA of E.coli.<br/>
| |
- | --Primers--<br/>
| |
- | glnL+G<br/>
| |
- | Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac<br/>
| |
- | Right primer: ggactagtacacacaagctgtgaatcactc<br/>
| |
- | rpoN<br/>
| |
- | Left primer: ggaattcgcggccgcttctagaggttctgaacatgaagcaa<br/>
| |
- | Right primer: ggactagtatccttatcggttgggtca<br/>
| |
- | annealing temperature was 56 degrees.<br/>
| |
- |
| |
- | [[File:Kyoto-Gel08300.jpg]]<br/>
| |
- | lane1: 100bp DNA ladder, lane2:glnL, lane3:glnG, lane4:glnG+L, lane5:rpoN from gDNA, lane6:rpoN from ASKA clone<br/>
| |
- |
| |
- | After purification, the concentration of DNA are<br/>
| |
- | glnL: 122.3 ng/ul<br/>
| |
- | glnG: 64.7 ng/ul<br/>
| |
- | glnL+G: 106.7 ng/ul<br/>
| |
- | rpoN from gDNA: 111.4 ng/ul<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・Screening PCR of σ54 promoter + pSB1A3<br/>
| |
- | [[File:Kyoto-Gel08301.jpg]]<br/>
| |
- | We cultured σ54 promoter5<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・Restriction of σ54 promoter5,glnL, glnG, glnL+G and rpoN<br/>
| |
- | Cut them with EcoRl and Spel
| |
- | After purification, the concentration of DNA were<br/>
| |
- | σ54 promoter5: 23.6 ng/ul<br/>
| |
- | glnL: 28.1 ng/ul<br/>
| |
- | glnG: 26.3 ng/ul<br/>
| |
- | glnL+G: 15.3 ng/ul<br/>
| |
- | rpoN: 20.8 ng/ul<br/>
| |
- |
| |
- | ・Ligation reaction<br/>
| |
- | Ligated glnL, glnG and rpoN to pSB1K3.<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・Screening PCR of glnL, glnG, glnL+G and rpoN<br/>
| |
- | glnL
| |
- | [[File:Kyoto-Gel09020.jpg]]<br/>
| |
- | glnG
| |
- | [[File:Kyoto-Gel09021.jpg]]<br/>
| |
- | glnL+G & rpoN
| |
- | [[File:Kyoto-Gel09022.jpg]]<br/>
| |
- | We cultured glnL5, glnG4<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・Mini prep of glnL5 and glnG4<br/>
| |
- | glnL5: 43.9 ng/ul<br/>
| |
- | glnG4: 38.1 ng/ul<br/>
| |
- |
| |
- | ・Screening PCR of rpoN
| |
- | [[File:Kyoto-Gel09020.jpg]]<br/>
| |
- |
| |
- | Predation(Hashiya)<br/>
| |
- | ・PCR amplification of glmS<br/>
| |
- | --Primers--<br/>
| |
- | left primer:ggaattcgcggccgcttctagagcaggttgaccgacaacgata<br/>
| |
- | right primer:ggactagtacgcagggcatccatttat<br/>
| |
- | [[File:Kyoto-Gel09030.jpg]]<br/>
| |
- | lane1: 100bp DNA ladder, lane2: glmS from gDNA, lane3: glmS from ASKA clone<br/>
| |
- |
| |
- | ・TA cloning of glmS<br/>
| |
- | Ligated glmS from gDNA to pTA vector.<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・Screening PCR of rpoN<br/>
| |
- | [[File:Kyoto-Gel09020.jpg]]<br/>
| |
- |
| |
- | ・Transformation of bellow parts<br/>
| |
- | 1-23L: BBa_B0015 (double terminator)<br/>
| |
- | 1-18E: BBa_J23101 (constitutive promoter)<br/>
| |
- | 1-18C: BBa_J23100 (constitutive promoter)<br/>
| |
- |
| |
- | <html><a name="lab-week6"></a></html>
| |
- |
| |
- | == Week6: Monday 5th September - Sunday 11th September ==
| |
- | '''Monday'''<br/>
| |
- | Nutritional Signal(Hashiya)<br/>
| |
- | ・Screening PCR of glnL+G+double terminator<br/>
| |
- |
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- | Luminescence(Kusaba, Hara):ハエの走行性実験②(②は改良版) ♂、緑×2回 ♀、青×1回
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- | Luminescence(Hara):ハエの走行性実験② ♂、青×2回 ♀、緑×2回、青×1回
| |
- |
| |
- | '''Thursday'''<br/>
| |
- | Luminescence(Hara):ハエの走行性実験② ♂、紫外線×3回 ♀、紫外線×3回
| |
- |
| |
- | '''Friday'''<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- | Luminescence(Kusaba, Hara):ハエの走行性実験② ♂、青×2回、赤×2回 ♀、青×2回、赤×2回
| |
- |
| |
- | '''Sunday'''<br/>
| |
- | Luminescence(Kusaba, Hara):ハエの走行性実験② ♂、紫外線×2回、
| |
- | <html><a name="lab-week7"></a></html>
| |
- |
| |
- | == Week7: Monday 12th September - Sunday 18th September ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | Luminescence:大腸菌の形質転換(Hashiya) ハエの走行性実験②(Kusaba, Hara)
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | Luminescence:大腸菌はじめて光る。しかし光量は少ない。
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- |
| |
- | Digestion(Kajita)
| |
- |
| |
- | PCR amplification of SAM-P20 and ChiA
| |
- |
| |
- | :We performed colony direct PCRs from a ''S. albogriseolus'' colony and a ''S. avermitilis'' colony.
| |
- |
| |
- | <table style="float:left;"><tr><td>
| |
- | :{|class="wikitable"
| |
- | |+Reaction mixture
| |
- | ! width="80"|Component
| |
- | !width="80"|Volume(μl)
| |
- | |-
| |
- | |2x Buffer||25
| |
- | |-
| |
- | |2mM dNTPs||10
| |
- | |-
| |
- | |Primer 1||1.5
| |
- | |-
| |
- | |Primer 2||1.5
| |
- | |-
| |
- | |Template||1
| |
- | |-
| |
- | |ddH<sub>2</sub>O||up to 50
| |
- | |}
| |
- | </td></tr></table>
| |
- | <table><tr><td>
| |
- | :{|class="wikitable"
| |
- | |+PCR condition
| |
- | |Predenature||94C||2m||
| |
- | |-
| |
- | |Denature||98C||10s||rowspan="3"|25cycles
| |
- | |-
| |
- | |Annealing||56C||30s
| |
- | |-
| |
- | |Extension||68C||1m30s
| |
- | |}
| |
- | </td></tr></table>
| |
- |
| |
- | <br style="clear: both;">
| |
- |
| |
- | '''Saturday'''<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="lab-week8"></a></html>
| |
- |
| |
- | == Week8: Monday 19th September - Sunday 25th September ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="lab-week9"></a></html>
| |
- | == Week9: Monday 26th September - Sunday 2nd October ==
| |
- | '''Monday'''<br/>
| |
- |
| |
- | '''Tuesday'''<br/>
| |
- |
| |
- | '''Wednesday'''<br/>
| |
- |
| |
- | '''Thursday'''<br/>
| |
- |
| |
- | '''Friday'''<br/>
| |
- |
| |
- | '''Saturday'''<br/>
| |
- |
| |
- | '''Sunday'''<br/>
| |
- |
| |
- | <html><a name="protocol"></a></html>
| |
- | == Protocol ==
| |
- |
| |
- | === Medium for drosophila ===
| |
- | ::{| class="wikitable"
| |
- | |+ [http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]
| |
- | ! width="300" |Materials
| |
- | ! width="300" |Methods
| |
- | |-
| |
- | |
| |
- | * water : 500mL
| |
- | * dry yeast : 20g
| |
- | * corn flour : 45g
| |
- | * glucose : 50g
| |
- | * agarose : 3.5~5g
| |
- | * propionic acid : 1.5mL
| |
- | * 10% p-hydroxybenzoate in 70% Eternol : 5g
| |
- | |
| |
- | # Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it.
| |
- | # Stir corn flour and glucose with the remaining water.
| |
- | # Stir 1 and 2, then autoclave it again.
| |
- | # after autoclave, add propionic acid and 10% p-hydroxybenzoate in 70% Eternol into it.■
| |
- | |}
| |
- |
| |
- |
| |
- |
| |
- |
| |
- |
| |
- |
| |
- | </div>
| |
- | <!-- /#main -->
| |