Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. Invitrogen lipofectamine transfection reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Thee two most important factors in choosing transfecting reagent are. Lipofectamine 2000 dna transfection reagent protocol. Higher densities can lead to spontaneous differentiation. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Add 6l of lipofectamine2000 from biostores and 100 l of. To transfect cells with sirna, follow the protocol as described for dna but. Highest transfection efficiency in many cell types and formats e. Using hek293t expression system to study photoactive. Plate cells for transfection only 1 day before the experiment. Transfection guide overview of transfection methods. Lipofectamine 2000 reagent protocol thermo fisher scientific.
Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. To transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells, and. For example, one reagent may work well with hek293 cells, but another. Hek293 cells exhibited the highest egfp expression compared to the bj. Lipofectamine 2000 transfection reagent from invitrogen. This is often the best place to start, especially in a new cell line. Transfection efficiencies lower than 50% were not reported in this table.
Transfection guide overview of transfection methods promega. Kit qiagen, hilden, germany according manufacturers instructions and the dna. However, i could only reach at most 2030% transfection efficiency. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Htm cells were transfected using lipofectamine with a fluorescent mirna dy547 and analyzed 48 hours after transfection. Optimized conditions for successful transfection of human. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Maintain p19 cells undifferentiated in mem with 10% serum 7. In a reverse transfection protocol, cells are added directly to a plate. Refer to the cell lines database for a list of cell types successfully.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. If you find this doesnt work for your specific cell type, then you can look to our cell specific protocols below for further optimization. Lipofectamine reagent thermo fisher and its newer derivatives are widely used for. Transfection of cells with different lipofectamine 2000 amounts. Lipofectamine 2000 is cationic based lipid delivery system, which transports dna into cells by fusing with plasma membrane. Invitrogen lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. The cells should not be old, preferably 1st to 4th passage is good for high transfection efficiency. I always use lipofectamine 2000 for transfection of hek293t cells. The hek293 cells cotransfected with cry2cib1 or cry2spa1 were.
The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. The day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. Gently mix media on cells via figure 8 pattern and back and forth motions. Plate cells so they will be 7090% confluent at the time of transfection. Can anyone provide me with a proper transfection protocol. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Lipofectamine 3000 reagent thermo fisher scientific us. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. Transfecting plasmid dna into hek 293 cells using lipofectamine. Lipofectamine 2000 is that it has an extensive, very useful manual. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Count the cells using standard trypan blue exclusion. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. Can anyone provide me with a proper transfection protocol into. According to the manufacture manual, pcr products were first mixed with 10.
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