What is the role of lipofectamine in transfection?
What is the role of lipofectamine in transfection?
It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the transfection payload, e.g. DNA plasmids.
How does cell type affect transfection efficiency?
Too high of a cell density can cause contact inhibition, resulting in poor uptake of nucleic acids and/or decreased expression of the transfected gene. Similarly, actively dividing cell lines are more efficiently transduced with viral vectors.
How confluent should cells be for transfection?
As a general guideline, transfect cells at 40–80% confluency. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells results in contact inhibition, making cells resistant to the uptake of foreign DNA. Actively dividing cells take up introduced DNA better than quiescent cells.
How long to transfect PC12 cells with Lipofectamine?
Return the cells to the 10% CO2 incubator. This protocol typical has given 1–2% transfected cells, as judged by staining with X-gal for a CMV-beta-galactosidase reporter gene. I usually let the cells grow for 40 to 60 h before harvesting in transient transfections.
Which is the best protocol for Lipofectamine transfection?
Note: Invitrogen Lipofectamine Transfection Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. This is often the best place to start, especially in a new cell line.
Where did the C2C12 cell line come from?
C2C12 cells were generated by Blau, Chiu, and Webster (1983) as a subclone of the C2 cell line isolated by Yaffe and Saxel (1977). C2C12 cells were originally derived from satellite cells from the thigh muscle of a 2-month-old female C3H mouse donor 70 h after a crush injury.
How is the differentiation of C2C12 cells achieved?
Differentiation of C2C12 cells is achieved by replacing GM to differentiation media, DM [DMEM—high glucose no sodium pyruvate (Gibco), 2% horse serum (Gibco), 1% glutamine (Gibco), 1% pen/strep (Gibco)]. After 24 h in DM, fused cells should be visible.