pPICZ-mEGFP-hPORCN

Protein Expression Eukaryotic cells - HEK293 mEGFP-hPORCN

Experiment
Protein Expression Eukaryotic cells - HEK293 mEGFP-hPORCN
Product
pPICZ-mEGFP-hPORCN from Anirban Banerjee, Cell Biology and Neurobiology Branch, National
Manufacturer
Anirban Banerjee, Cell Biology and Neurobiology Branch, National

Protocol tips

Protocol tips
The cells were transiently transfected using polyethyleneimine (PEI) [55]. The evening before the day of transfection, cells from T-75 flasks or 150 mm cell culture dishes were passaged and ~ 0.25 ×106 cells seeded into 12-well plates with 1 ml media per well. The next morning, 1 μg DNA was diluted into 50 μl of DMEM media. A separate tube contained 3 ug of PEI diluted into 50 μl of DMEM media.

Publication protocol

HEK293T cells were maintained in DMEM media supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin/streptomycin in a humidified incubator at 37°C with 5% CO2. The cells were transiently transfected using polyethyleneimine (PEI) [55]. The evening before the day of transfection, cells from T-75 flasks or 150 mm cell culture dishes were passaged and ~ 0.25 ×106 cells seeded into 12-well plates with 1 ml media per well. The next morning, 1 μg DNA was diluted into 50 μl of DMEM media. A separate tube contained 3 ug of PEI diluted into 50 μl of DMEM media. The two solutions were mixed, pipetted, and incubated for 15–20 min at room temperature. Then ~ 100 μl per well of the DNA:PEI complex was added dropwise to the wells, the plate gently agitated and returned to the 37°C incubator. Alternatively, post addition of the DNA:PEI complex, the plate was incubated in a humidified shaker maintained at 30°C with 5% CO2 and the shaking turned off. For hPORCN constructs, we obtained more consistent results in doing the transient transfections in 6-well plates seeded with ~0.75×106 cells and transfected using 2 μg DNA complexed with 6 μg PEI per well. Larger scale transfections for protein purifications were done in 150-mm cell culture dishes. The dishes were initially seeded with ~10×106 cells and transfected the next day with 30 μg DNA complexed with 90 μg PEI in a final volume of 2 ml DMEM media.

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