Transfection

MDS offers a range of services that enable sensitive and accurate determination of gene expression levels. The outline of transfection and purification process that we follow is summarized below.

Vector construction: The desired cDNA is cloned into a mammalian expression vector that carries the gene for neomycin resistance. The construct is fully sequence verified and a large-scale preparation of plasmid construct DNA is made.

Transfection: The construct is incubated with a transfection reagent and transfected into a host cell, typically COS-1, CHO KI or HEK 293 cells. Cotransfection of the vector services are also available. The services include:

Electroporation
• Adherent Cell
• Suspension Cells
Lipofection Transfection
• Adherent Cells
• Suspension Cells

Insert confirmation: Verification of the transfer of the desired insert into the vector is performed by PCR analysis. Verification of the expression with the client: Both cells and supernatant from plaque-purified recombinants are sent to the client for analysis. We recommend Western or ELISA assays to determine if the protein of interest is present.

Selection and Enrichment: Cells that carry the construct are selected using neomycin resistance carried by the expression vector. Clones expressing the target protein are selected using Quantitative PCR and/or Western blotting. Clonal selection is performed by dilution in 96-well plates and subsequent propagation of clones of interest.

Deliverables: A final cell line with the desired characteristics is selected. Mycoplasma testing is carried out on all clones prior to shipping worldwide.

Additional Services: MDS's Technical Services Division offers many additional services to save you valuable time and resources. Additional services include:

• Purification of expressed protein (yields may be improved through addition of affinity tags) Analysis of expressed protein by SDS-PAGE and Western blot (utilizing your specific antibody).
• Filling, capping, and labeling services are also available. Pricing is based on the complexity of each project, the type of cell selected, and the services rendered. MDS's custom protein expression, purification, and characterization services can be tailored to suit your needs.