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Transfection of S2 Cell with DNA Using CellFectin Reagent
Published: Mar 5, 2012 DOI: 10.21769/BioProtoc.190 Views: 22303
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Abstract
This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition of copper sulfate, which can then be purified and used as desired. This protocol provides an example to finding out when your peak protein production occurs and a method for determining optimal selection conditions.
Materials and Reagents
- Drosophila Schneider 2 (S2) cells (Life Technologies, Invitrogen™, catalog number: R690-07 )
- Dros-SFM (Life Technologies, Invitrogen™, catalog number: 10797-025 )
- pHASβAG (pRmHA-3 vector with SDF-1β-FLAG inserted. Contains a metallothionein promoter inducible with Cu2+)
- pUC-Hsneo (contains the neomycin-resistant cassette required for stable selection)
- CellFectin reagent (Life Technologies, Invitrogen™, catalog number: 10362-010 )
- FBS (Life Technologies, Invitrogen™, catalog number: 10437-028 )
- Penicillin-Streptomycin (P/S) (Life Technologies, Invitrogen™, catalog number: 15140-122 )
- G418 sulphate (Life Technologies, Invitrogen™, catalog number: 11811-031 )
- CuSO4 (Sigma Aldrich, catalog number: C8027 )
- HT supplement (Life Technologies, Invitrogen™, catalog number: 11067-030 )
- Transfection solutions (see Recipes)
Equipment
- 6-well plate (Corning Incorporated, catalog number: 353046 )
- 24-well plate (Corning Incorporated, catalog number: 353047 )
Procedure
Category
Molecular Biology > DNA > Transformation
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