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Team:ZJU-China/labnote8.htm
From 2012.igem.org
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<p align="justify">1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.</p> | <p align="justify">1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.</p> | ||
<p align="justify">2. PCR of part K738001:</p> | <p align="justify">2. PCR of part K738001:</p> | ||
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/0/0a/Zju_notebook19.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/0/0a/Zju_notebook19.jpg" width="500px"><p align="justify"></div> |
<p align="justify">3. PCR of original pCJDFA and pCJDFB.</p> | <p align="justify">3. PCR of original pCJDFA and pCJDFB.</p> | ||
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/f/f3/Zju_notebook20.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/f/f3/Zju_notebook20.jpg" width="500px"><p align="justify"></div> |
<p align="justify">4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.</p> | <p align="justify">4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.</p> | ||
<p align="justify">Name: FAM-8-6-1 Concentration: 56.7 ng/ul A260/280: 1.95</p> | <p align="justify">Name: FAM-8-6-1 Concentration: 56.7 ng/ul A260/280: 1.95</p> | ||
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<p align="justify">PCR1: delete 3bp after MS2 in D0.</p> | <p align="justify">PCR1: delete 3bp after MS2 in D0.</p> | ||
<p align="justify">PCR2: delete 3bp before MS2 in D0.</p> | <p align="justify">PCR2: delete 3bp before MS2 in D0.</p> | ||
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/9/94/Zju_notebook21.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/9/94/Zju_notebook21.jpg" width="500px"><p align="justify"></div> |
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/b/bb/Zju_notebook22.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/b/bb/Zju_notebook22.jpg" width="500px"><p align="justify"></div> |
<h3>August 10</h3> | <h3>August 10</h3> | ||
<p align="justify">1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.</p> | <p align="justify">1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.</p> | ||
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<p align="justify">3. Mutation of D0: </p> | <p align="justify">3. Mutation of D0: </p> | ||
<p align="justify">PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.</p> | <p align="justify">PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.</p> | ||
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/7/7c/Zju_notebook23.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/7/7c/Zju_notebook23.jpg" width="500px"><p align="justify"></div> |
<p align="justify">PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.</p> | <p align="justify">PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.</p> | ||
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/6/63/Zju_notebook24.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/6/63/Zju_notebook24.jpg" width="500px"><p align="justify"></div> |
<p align="justify">PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.</p> | <p align="justify">PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.</p> | ||
| - | <p align="justify"><img src="https://static.igem.org/mediawiki/igem.org/7/7d/Zju_notebook25.jpg" width="500px"><p align="justify"> | + | <p align="justify"><div class="floatC"><img src="https://static.igem.org/mediawiki/igem.org/7/7d/Zju_notebook25.jpg" width="500px"><p align="justify"></div> |
<p align="justify">4. Amplify FB-8-7 in LB liquid medium.</p> | <p align="justify">4. Amplify FB-8-7 in LB liquid medium.</p> | ||
Latest revision as of 17:19, 26 October 2012
Week 8 (08.06-08.12):
August 06
1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.
2. PCR of part K738001:
3. PCR of original pCJDFA and pCJDFB.
4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.
Name: FAM-8-6-1 Concentration: 56.7 ng/ul A260/280: 1.95
Name: FAM-8-6-2 Concentration: 57.3 ng/ul A260/280: 1.95
Name: FAM-8-6-3 Concentration: 86.8 ng/ul A260/280: 2.06
Name: FBP-8-6-1 Concentration: 21.0 ng/ul A260/280: 2.14
Name: FBP-8-6-2 Concentration: 15.5 ng/ul A260/280: 2.14
Name: FBP-8-6-3 Concentration: 15.6 ng/ul A260/280: 2.07
5. Add theophylline 0.08g/10mL into Venus-8-6, observe through fluorescence microscope.
6. Purify PCR product of K738001, named K738001-8-6.
7. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3), named Co3-8-6. Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3), named Co2-8-6.
August 07
1. Double digest K738001-8-6 with Xba1 and Spe1. Purify the product before ligation step.
2. Ligation: K738001-8-6 and pSB1C3.
3. Transform the product of ligation into DH5α competent cells.
August 08
Pick up single colonies from plate of K738001-8-7 and amplify it in LB liquid medium and LB plate.
August 09
1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium, induce with IPTG when OD600 is about 0.6 (mid log stage).
2. Miniprep plasmids of K738001-8-8:
Name: K738001-8-9-1 Concentration: 174.2 ng/ul A260/280: 1.91
Name: K738001-8-9-2 Concentration: 298.5 ng/ul A260/280: 1.87
3. Amplify FBP overnight for minipreparing plasmids later.
4. Mutation of D0:
PCR1: delete 3bp after MS2 in D0.
PCR2: delete 3bp before MS2 in D0.
August 10
1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.
2. Miniprep plasmids of FB-8-9.
Name: FB-8-10-1 Concentration: 14.6 ng/ul A260/280: 1.97
Name: FB-8-10-2 Concentration: 24.4 ng/ul A260/280: 1.93
3. Mutation of D0:
PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.
PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.
PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.
4. Amplify FB-8-7 in LB liquid medium.
August 11
1. Miniprep plasmids of FB-8-11.
Name: FB-8-11-1 Concentration: 33.9 ng/ul A260/280: 2.06
Name: FB-8-11-2 Concentration: 50.2 ng/ul A260/280: 2.04
2. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Test fluorescence strength with hybrid synergy reader. Exciting light 535nm, absorbing light 480nm.
