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BACKGROUND
Transient transfection method introduces foreign nucleic acids (DNA) into cells to produce genetically altered cells for a defined period of time and an essential tool for introducing a gene function and regulation and translated protein functionality.
The purpose of this experiment is to study about transient transfection of the gene in a cell line.
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REQUIREMENTS
Sample: HEK293 cell line, gene of interest
Apparatus: Tissue culture level 24-well plates,
1.5 ml Eppendorf tube.
Chemicals: Culture media (DMEM, serum,
Antibiotics/Antimycotics),
L-glutamine,
Lipofectamine™ LTX Reagent,
Opti-MEM® Ι reduced serum media.
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PROCEDURE
Harvest HEK293 cells 48 hours before transfection. Cells should be 70% confluent for the transfection. Prior to start transfection, wash cells with the culture media and add transfection solution (0.5µg of DNA in Opti-MEM® Ι reduced serum media [100µl]1) further add 1.0-1.5 µl of Lipofectamine LTX™ Reagent into the transfection solution, gently mis and incubate 30 minutes at RT to generate a complexes of DNA- Lipofectamine LTX™ Reagent. After incubation, introduce 100 µl of the DNA- Lipofectamine LTX™ Reagent complexes straight to the well containing no. of cells and mix homogeneously by flip-flop the plate. 24 hours incubation, post-transfection before assaying for transgene expression.2
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CONCLUSION
Author concluded the transient transfection assay with Lipofectamine LTXTM reagent.
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REFERENCES
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Transfecting Plasmid DNA into HEK 293 Cells Using Lipofectamine™ LTX Reagent.
