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Construction and Packaging Services of AAV

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Transient Transfection Protocol
Transfection is a method to introduce nucleic acid into eukaryotic cells, and then proteins could be produced. The conventional transfection techniques include transient transfection and stable transfection. Transient transfection means that the constructed plasmids are introduced into eukaryotic cells only for a limited period of time and is not integrated into the genome. The foreign genes will be gradually lost during cell growth and division. However, the high copy number of the transfected genetic material can realize high levels of expressed protein within the period that it exists in the cell. So, transient transfection has been widely used for rapid expression protein with high activity in the short term. While, stable transfection can produce large amounts of protein in the long-term by constructing stable cell lines.

The Protocol of transient Transfection is as follow.
Cell recovery
Take out the cell from the liquid nitrogen or refrigerator, place it quickly in the preheated water bath ( 37°C), and slowly shake the frozen pipes until liquid is completely melted. The key of cell recovery is fast which is to prevent the cell damages during thawing.
Use a pipette to gently add the liquid into a culturing bottle with culture medium, place it in CO2 incubator, 37℃.
When the cell is attached to the wall, discard the supernatant and replace with a fresh culture medium. Then the cell could be cultivated by the conventional method.
The cultured cells are inoculated into 6-wells plates. Add 2-3 mL medium, mix and place in CO2 incubator, 37°C for the night. When around 70%-80% unilaminar cells formed at the bottom of culture flask, wash cells  with serum-free medium and add 1 mL serum-free medium each well. 2 μg plasmid is diluted with 100 μL serum-free medium, fully mix, and then add  transfection reagent, mix well again. let rest about 15 min at room temperature. Then add the solution to each well in drops, mix gently place in COincubator, 37°C for 24 hours.
Expression detection
Collect cultured cells, break up the cells through ultrasound or enzymatic hydrolysis, and supernatant is obtained by centrifugation. The detection of mRNA expression level and protein expression level are conducted. The cDNA need analyses with real time-PCR or reverse transcription PCR. And proteins are extracted for western blot analysis.

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