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Team:Freiburg/Content/Notebook/Labjournal
From 2014.igem.org
(Difference between revisions)
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<h2 id="Viral-Vectors-May">Viral Vectors - May</h2> | <h2 id="Viral-Vectors-May">Viral Vectors - May</h2> | ||
| + | <h3>2014/05/21</h3> | ||
| + | <h4>Transfection/ Virus production</h4> | ||
| + | <p>For virus production Phoenix cells (producer cell line) were splitted (well separated) on 100mm plates. At 70% cell density cells were transfected using polyethylenimine.</p> | ||
| + | <ul> | ||
| + | <li>remove medium and refill with 5 ml new completed growth medium (DMEM)</li> | ||
| + | <li>600 µl transfection mastermix was prepared (8 µg pMIG IRES EGFP, 24 µl PEI, rest OptiMEM)</li> | ||
| + | <li>mastermix was incubated 15 min and carefully drop on the plates</li> | ||
| + | </ul> | ||
| + | <p>Plates were incubated at 37°C. The supernatant after 24 was removed and refilled with 5 ml new DMEM, the supernatant was collected after 48 h (refilled with 5 ml DMEM) as well as 72 h.</p> | ||
| + | <figure> | ||
| + | <img src="" alt="Description of Image"> | ||
| + | <figcaption> | ||
| + | <p class="header">??</p> | ||
| + | <p class="desc">Phoenix cells transfected with pMIG IRES EGFP one day after transfection (2014-05-22-phoenix-100mm-pmig-ires-egfp-8µl-transf-24h)</p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | |||
| + | <h3>2014/05/25</h3> | ||
| + | <h4>Transduction mouse cells</h4> | ||
| + | <p>NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP.</p> | ||
| + | <figure> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/2/29/Freiburg_Notebook_2014_5_14_bild1.png | ||
| + | " alt="Description of Image"> | ||
| + | <figcaption> | ||
| + | <p class="header">???</p> | ||
| + | <p class="desc">???</p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | <ul> | ||
| + | <li>500 µl of supernatant was removed</li> | ||
| + | <li>1 µl Polybrene was added (10mg/ml)</li> | ||
| + | <li>500 µl virus supernatant was added (sterile filtered)</li> | ||
| + | <li>incubation at 37°C for 6h</li> | ||
| + | <li>cell supernatant was replaced with fresh DMEM</li> | ||
| + | <li>transduction was repeated once</li> | ||
| + | </ul> | ||
| + | <p>Pictures could be made after 48 h of incubation.</p> | ||
| + | <figure> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/2/29/Freiburg_Notebook_2014_5_14_bild1.png" alt="Description of Image"> | ||
| + | <figcaption> | ||
| + | <p class="header">Anderes Foto</p> | ||
| + | <p class="desc">NIH 3T3 cells were transduced twice with MuLV IRES EGFP for 6h. Picture was made after 48 h of incubation at 37°C (2014-05-28-nih-3t3-100mm-mulv-pmig-ires-egfp-transd-48h)</p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | |||
<h2 id="Viral-Vectors-June">Viral Vectors - June</h2> | <h2 id="Viral-Vectors-June">Viral Vectors - June</h2> | ||
Revision as of 13:15, 3 October 2014
Cloning
Cloning - May
Cloning - June
Cloning - July
Cloning - August
Cloning - September
Cloning - October
Viral Vectors
Viral Vectors - May
2014/05/21
Transfection/ Virus production
For virus production Phoenix cells (producer cell line) were splitted (well separated) on 100mm plates. At 70% cell density cells were transfected using polyethylenimine.
- remove medium and refill with 5 ml new completed growth medium (DMEM)
- 600 µl transfection mastermix was prepared (8 µg pMIG IRES EGFP, 24 µl PEI, rest OptiMEM)
- mastermix was incubated 15 min and carefully drop on the plates
Plates were incubated at 37°C. The supernatant after 24 was removed and refilled with 5 ml new DMEM, the supernatant was collected after 48 h (refilled with 5 ml DMEM) as well as 72 h.
??
Phoenix cells transfected with pMIG IRES EGFP one day after transfection (2014-05-22-phoenix-100mm-pmig-ires-egfp-8µl-transf-24h)
2014/05/25
Transduction mouse cells
NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP.
???
???
- 500 µl of supernatant was removed
- 1 µl Polybrene was added (10mg/ml)
- 500 µl virus supernatant was added (sterile filtered)
- incubation at 37°C for 6h
- cell supernatant was replaced with fresh DMEM
- transduction was repeated once
Pictures could be made after 48 h of incubation.
Anderes Foto
NIH 3T3 cells were transduced twice with MuLV IRES EGFP for 6h. Picture was made after 48 h of incubation at 37°C (2014-05-28-nih-3t3-100mm-mulv-pmig-ires-egfp-transd-48h)
