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This is intended as a guide only; for full experimental details please read the reference provided.
Sufficient plasmid DNA must first be generated. Begin by amplifying the quantity of the plasmid by performing bacterial transformation. Grow the transformed bacterial culture overnight (in 37°C shaker) and extract the plasmid DNA using DNA MAXIprep kit.
Culture HEK293T cells in HEK media (DMEM/F-12 media, FBS, non-essential amino acids, L-glutamine) within humidified incubators (standard conditions of 37˚C, 5% CO2 and 21% O2). Once the culture has reached high confluency (>90%), the cells are ready for transfection.
Polybrene (Cat. No. 7711) is supplied as a 10 mg solid and is soluble in water (up to 100 mg/mL). To generate a working solution, Polybrene should first be dissolved in 1 mL sterile water to generate a 10 mg/mL stock solution.
For more information on preparing stock solutions, see our Molarity Calculator.
|Stock solution Polybrene concentration (mg/mL)||Stock volume (mL)||Stock Polybrene (Solid, mg)||Sterile Ultra-Pure Water (mL)|
Polybrene working solution is generated from the polybrene stock solution (10 mg/mL in sterile water). This stock solution can be further diluted in the culture media to generate the desired working concentration for the transfection or transduction media. Example dilutions shown below.
For more information on calculating dilutions see our Dilution Calculator.
|Final desired working concentration (µg/mL)||Dilution||Stock solution Polybrene concentration (mg/mL)||Volume of stock solution Polybrene (µL)||Volume of culture media (mL)|