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Team:Freiburg/Content/Notebook/Labjournal
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<Section id="Viral-Vectors"> | <Section id="Viral-Vectors"> | ||
<h1>Viral Vectors</h1> | <h1>Viral Vectors</h1> | ||
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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> | + | <h3>2014/05/21</h3> |
| - | <h4>Transfection/ Virus production</h4> | + | |
| - | + | <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="https://static.igem.org/mediawiki/2014/c/ca/Freiburg_Notebook_2014-05-22-phoenix-100mm-pmig-ires-egfp-8%C2%B5l-transf-24h.jpg" alt="Description of Image"> | |
| - | + | <figcaption> | |
| - | + | <p class="desc">Phoenix cells transfected with pMIG IRES EGFP one day after transfection.</p> | |
| - | + | </figcaption> | |
| - | + | </figure> | |
| + | |||
| + | <h3>2014/05/25</h3> | ||
| - | + | <h4>Transduction mouse cells</h4> | |
| - | <h4>Transduction mouse cells</h4> | + | <div class="row category-row"> |
| - | + | <div class="col-sm-6"> | |
| - | + | <p>NIH 3T3 cells (60% density) were transduced with MuLV IRES EGFP.</p> | |
| - | + | <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> | |
| - | + | </div> | |
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| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
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| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | </ | + | |
| - | + | ||
| + | <div class="col-sm-6"> | ||
| + | <figure> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/2/29/Freiburg_Notebook_2014_5_14_bild1.png | ||
| + | " alt="Description of Image"> | ||
| + | </figure> | ||
| + | |||
| + | <figure> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/1/10/Freiburg_Notebook_2014-05-28-nih-3t3-100mm-mulv-pmig-ires-egfp-transd-48h.tif" alt="Description of Image"> | ||
| + | <figcaption> | ||
| + | <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.</p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | |||
<h2 id="Viral-Vectors-June">Viral Vectors - June</h2> | <h2 id="Viral-Vectors-June">Viral Vectors - June</h2> | ||
Revision as of 14:46, 11 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.
2014/05/25
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.
NIH 3T3 cells were transduced twice with MuLV IRES EGFP for 6h. Picture was made after 48 h of incubation at 37°C.
Viral Vectors - June
2014/06/20
Thawing of eukaryotic cells
New Phoenix cell stocks were thawed:
- cryotube was thawed at 37°C water bath until almost defrosted
- stock was filled in 9 ml warm DMEM and centrifuged at 900 rpm for 2 min
- medium was removed and refilled with 10 ml warm DMEM
- cells were seeded on 100 mm plate
Testing optimal cell density of mouse fibroblasts
NIH 3T3 have a really fast growth so that we tested the optimal cell number for seeding NIH 3T3 for having around 60% cell density on the next day. NIH 3T3 cells grow very fast; therefore we have tested the optimal seeding cell number to obtain 60% cell density on the next day.
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)
- incubation for 24 h at 37°C
--> optimal cell number is 1 – 1.5x10^5 cells per well ( = 0.5 – 0.75 cells/ml)
2014/06/22
Transfection/ Virus production
Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (2 x 100mm plate)
Anderes Foto
3 Phoenix cells transfected with pMIG IRES EGFP after 3d.
2014/06/24
Transfection/ Virus production
Transfection of Phoenix cells (70% density) with pMIG IRES EGFP (protocol: 2014/05/21) (5 x 100mm plate)
2014/06/27
Thawing new HEK 293 cells
(protocol: 2014/06/20)
Transfection CHO cells with receptor
Transfection of CHO cells with SLC7a1 (for later transduction with virus). Medium was changed after 5 h. Cells were incubated for 24 h before viral transduction with MuLV IRES EGFP, medium change after 16 h.
200 µl transfection mix per well (3 µg DNA, 7,5 µl PEI, rest OptiMEM), transfection control: pEGFP_C1
(left) Cho cells: without receptor were transduced with MuLV IRES EGFP ,(right) CHO cells transfected with SLC7a1 and transduced with MuLV IRES EGFP (24h after transfection)
