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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: None
Forgot to insert protocol.
Transfection Protocol -> CHO-DG44
- Cell Density 3x10^6cells/mL
- Total Volume 5 mL
- DNA 1.5 µg/10^6cells
- PEI 3 µg/10^6cells
- Passage seed 1 day prior to transfection
- Prepare tubes by addition of DNA
- Centrifuge the necessary volume of seed, remove conditioned medium and resuspend in necessary volume of fresh medium to acheive the required cell density
- Add 5 mL of the cell suspension to the tube with DNA and mix by orbitally shaking
- Add the PEI to the Cell+DNA mixture as soon as possible
- 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
