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Transfection of CHO cells
Protocol: Transfection of CHO cells
This is the transfection protocol used at the LBTC lab for CHO DG44 cells. The transfection reagent is PEI ([http://en.wikipedia.org/wiki/Polyethylenimine polyethylenimine]).
Please use the provided Excel sheet to calculate the volume of plasmid you should add to the cells. Replace every value in red by your own, then print the sheet out and follow the provided protocol.
1. Passage seed 1 day prior to transfection.
2. Prepare tubes (yellow caps with holes) by addition of the calculated amount of DNA.
3. Centrifuge the necessary volume of seed (after a PCV measurement), remove conditioned medium with the pump (use a 2 ml serological pipet with a broken neck) and resuspend (first in 10 ml) in necessary volume of fresh medium to achieve the required cell density (3 mio/ml).
4. Add 5 mL of the cell suspension to the tube with DNA and mix orbitally.
5. Add the PEI (45 µl) to the Cell+DNA mixture as soon as possible, flick 3 times.
6. Place in the incubator at 37°C.
We made 10 tubes with transfected CHO cells.
N° LovTAP dsRed Filler GFP pHY42 unit 1, 2 95 0 0 5 0 % 3 0 90 10 0 0 % 4 0 50 50 0 0 % 5 0 10 90 0 0 % 6 90 10 0 0 0 % 7 50 50 0 0 0 % 8 10 90 0 0 0 % 9, 10 0 0 0 0 100 %
The LovTAP ones were intended for Western blot, silver staining, FACS and microscopy, and the melanopsin ones for calcium imaging.
Transfection of HEK cells
Protocol: Transfection of HEK cells
This is the transfection protocol used at the LBTC lab for HEK cells. The transfection reagent is PEI ([http://en.wikipedia.org/wiki/Polyethylenimine polyethylenimine]).
Please use the provided Excel sheet to calculate the volume of plasmid you should add to the cells. Replace every value in red by your own, then print the sheet out and follow the provided protocol.
The difference with CHO cells is that a different cell density is required for transfection. You also use two different mediums (one for transfection and one for growth) instead of only one.
1. Passage seed 1 day prior to transfection.
2. Prepare tubes by addition of calculated volume of DNA as a droplet at the bottom.
3. Centrifuge (1500 rpm, 3 min) the necessary volume of seed, remove conditioned medium with the pump (use a 2 ml serological pipet with a broken neck) and resuspend in necessary volume of fresh transfection medium (RPMI) to acheive the required cell density (20 mio/ml).
4. Add 0.5 mL of the cell suspension to the tube with DNA and mix orbitally.
5. Add the PEI (30 ug) to the Cell+DNA mixture as soon as possible, flick 3 times.
6. Place in the incubator at 37°C.
7. Dilute the transfections with 9.5mL of growth medium (EX-CELL 293) 3 hours after the transfection.
Two tubes were transfected with melanopsin (100%).
All of the tubes were stored in the dark, in tubes covered with tape.
