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CHO cell transfection with LOVTAP and RO
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.
- CHO cells transfection with LovTAP and RO to see if some expression can be detected
- Culture of seed CHO cells (will be continued on 19th of July)
Transfection plan: different DNA percentages. Each one of those will have a duplicate. Made the Excel we use for our protocol, calculated total DNA with it. Here is the detail of the tubes we made:
DNA concentration (Stock values) :
| LovTAP | RO | filler | unit |
| 0.4 | 0.29 | 1.06 | µg/µL |
Percentage of DNA
| Tube number | LovTAP | RO | filler | unit |
| 1,2 | 100 | 0 | 0 | % |
| 3,4 | 10 | 0 | 90 | % |
| 5,6 | 0 | 0 | 100 | % |
| 7,8 | 50 | 50 | 0 | % |
| 9,10 | 0 | 100 | 0 | % |
| 11,12 | 0 | 50 | 50 | % |
Mass of DNA
| Tube number | LovTAP | RO | filler | unit |
| 1,2 | 22.5 | 0 | 0 | µg |
| 3,4 | 2.25 | 0 | 20.25 | µg |
| 5,6 | 0 | 0 | 22.5 | µg |
| 7,8 | 11.25 | 11.25 | 0 | µg |
| 9,10 | 0 | 22.5 | 0 | µg |
| 11,12 | 0 | 11.25 | 11.25 | µg |
Final volumes added :
| Tube number | LovTAP | RO | filler | unit |
| 1,2 | 56.25 | 0 | 0 | µL |
| 3,4 | 5.625 | 0 | 18.75 | µL |
| 5,6 | 0 | 0 | 20.8 | µL |
| 7,8 | 28.125 | 38.8 | 0 | µL |
| 9,10 | 0 | 77.59 | 0 | µL |
| 11,12 | 0 | 38.8 | 10.4 | µL |
Resuspension:
PCN found for 200µL of cell solution : 1.58 PCN for 100µL = 1.58/2 = 0.79
We need a PCN of 0.2 for 1mio cells/mL -> 4mio cells/mL for 0.79 PCN. Therefore we needed to add 49mL of new medium.
