Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Transfection of S2 Cell with DNA Using CellFectin Reagent

DC Darran G. Cronshaw

Published: Mar 5, 2012 DOI: 10.21769/BioProtoc.190 Views: 22250

download pdf Download PDF
ask Ask a question
Favorite
Cited by

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

  1. Drosophila Schneider 2 (S2) cells (Life Technologies, Invitrogen™, catalog number: R690-07 )
  2. Dros-SFM (Life Technologies, Invitrogen™, catalog number: 10797-025 )
  3. pHASβAG (pRmHA-3 vector with SDF-1β-FLAG inserted. Contains a metallothionein promoter inducible with Cu2+)
  4. pUC-Hsneo (contains the neomycin-resistant cassette required for stable selection)
  5. CellFectin reagent (Life Technologies, Invitrogen™, catalog number: 10362-010 )
  6. FBS (Life Technologies, Invitrogen™, catalog number: 10437-028 )
  7. Penicillin-Streptomycin (P/S) (Life Technologies, Invitrogen™, catalog number: 15140-122 )
  8. G418 sulphate (Life Technologies, Invitrogen™, catalog number: 11811-031 )
  9. CuSO4 (Sigma Aldrich, catalog number: C8027 )
  10. HT supplement (Life Technologies, Invitrogen™, catalog number: 11067-030 )
  11. Transfection solutions (see Recipes)

Equipment

  1. 6-well plate (Corning Incorporated, catalog number: 353046 )
  2. 24-well plate (Corning Incorporated, catalog number: 353047 )

Procedure

Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.

Category

Molecular Biology > DNA > Transformation

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A