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Lentivirus and Retrovirus Transfection
Published: Mar 5, 2011 DOI: 10.21769/BioProtoc.38 Views: 41624
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Abstract
293T and Pheonix cells grow in DMEM + 10%FBS. If you are transfecting other cells, you can use whatever medium those cells normally grow in and change to DMEM + 10%FBS on the day of the transfection. You can change back to “normal” medium 24 hours post-transfection. Calcium Phosphate transfection of M2182 in RPMI (w/o FBS) was reported to cause cells to die. All the transfection have been done in DMEM + 10% FBS, no transfection in other media was performed so far.
Materials and Reagents
- 293T and Pheonix cell line
- 1x Dulbecco's modified eagle medium (DMEM) (high glucose, liquid) (Life Technologies, InvitrogenTM)
- Fetal bovine serum (FBS) (Hyclone)
- Na2HPO4
- NaCl
- TE
- HEPES (Sigma-Aldrich, catalog number: H7006 )
- Packaging vector PcmvR8.74
- Envelop vector PMD2VSVG
- Reporter vector DsRED, GFP or LacZ
- 2x HBS (see Recipes)
- 2 M CaCl2 (Covidien, MallinkrodtTM, catalog number: 4160) (see Recipes)
Equipment
- Tissue culture incubator
- Tissue culture plates of the appropriate size (Thermo Fisher Scientific)
- 6 cm or 6 well plates
- Microscope
- 0.45 μm filter
- p1000 pipettor
Procedure
Category
Molecular Biology > DNA > Transfection
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