"
CHO cell transfection with LOVTAP and RO
- CHO cell transfection with LOVTAP and RO
- CHO cell new culturing (Will be continued on 19th of July)
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.
Transfection plan: different DNA percentages. Each one of those will have a duplicate.
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.
- Comments
We need to make a protocol page for CHO transfection
