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Team:Freiburg Bioware/NoteBook/Labjournal
From 2010.igem.org
(Difference between revisions)
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<h2 style="margin-left: 5px;">April</h2> | <h2 style="margin-left: 5px;">April</h2> | ||
<p class="boxes_labjournal_day">Labday 2- 5</p> | <p class="boxes_labjournal_day">Labday 2- 5</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: | + | <img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: |
| - | + | Vector plasmid; Thymidine kinase ;Cytosindeaminase" /> | |
| - | + | ||
| - | + | ||
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p> | <p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p> | ||
</a> | </a> | ||
| Line 81: | Line 79: | ||
<p class="boxes_labjournal_day">Labday 18- 45</p> | <p class="boxes_labjournal_day">Labday 18- 45</p> | ||
<img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title=" | <img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title=" | ||
| - | We conducted our first site-directed mutagenesis. | + | We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" /> |
| - | First cell culture steps: | + | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. | + | |
| - | + | ||
| - | Theoretical studies of the AAV structure: Impressions" /> | + | |
<p class="boxes_labjournal_text">...first transduced cells...</p> | <p class="boxes_labjournal_text">...first transduced cells...</p> | ||
</a> | </a> | ||
| Line 100: | Line 89: | ||
<p class="boxes_labjournal_day">Labday 46- 75</p> | <p class="boxes_labjournal_day">Labday 46- 75</p> | ||
<img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid. | <img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid. | ||
| - | For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI | + | For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." /> |
| - | + | ||
| - | BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. | + | |
| - | + | ||
| - | Further on we received first quantitative transduction results via flow cytometry. | + | |
| - | + | ||
| - | mGMK_TK30 was successfully cloned into the vector plasmid." /> | + | |
<p class="boxes_labjournal_text">...organizing the lab...</p> | <p class="boxes_labjournal_text">...organizing the lab...</p> | ||
</a> | </a> | ||
Revision as of 19:11, 27 October 2010
Get to know our virus
Our project starts with first sequence analysis.
Checking iGEM compatibility
Scanning for iGEM restriction sites:• Vector plasmid
• Thymidine kinase
• Cytosindeaminase
First practical steps
We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt.
First fluorescing HT1080 cells
We conducted our first site-directed mutagenesis.First cell culture steps:
• Splitting and seeding of AAV 293 and HT1080 cells
• Calcium phosphate transfection
• Transduction with virus particles containing YFP encoding sequence
• Microscopy: Fluorescent transduced HT1080 cells
We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared.
Theoretical studies of the AAV structure: Impressions.
Modifying Rep/Cap & Modularizing Vector plasmid
We developed a plan for modifying the surface exposed loops of the virus capsid. For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalIBioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix.
Further on we received first quantitative transduction results via flow cytometry.
mGMK_TK30 was successfully cloned into the vector plasmid.
Producing more BioBricks
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https://2010.igem.org/Team:Freiburg_Bioware/NoteBook => Back to Notebook overview]
- March (labday 1)
- April (labday 2 - 5)
- May (labday 6 - 17)
- June (labday 18 - 45)
- July (labday 46 - 75)
- August part 1 (labday 76 - 92)
- August part 2 (labday 93 - 106)
- September part 1 (labday 107 - 123)
- September part 2 (labday 124 - 135)
- October part 1 (labday 136 - 149 )
- October part 2 (labday 150 - 166 )
- November (labday 167 - 170 )
- Cellculture
