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Team:Freiburg/Content/Notebook/Labjournal
From 2014.igem.org
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.
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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.
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- 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)
