Team:EPF-Lausanne/Notebook/17 July 2012

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== CHO cell transfection with LOVTAP and RO ==
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== CHO cell transfection with LovTAP and RO ==
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* CHO cell transfection with LOVTAP and RO
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{{:Team:EPF-Lausanne/Template/Protocol|TransfectionCHO}}
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* CHO cell new culturing (Will be continued on 19th of July)
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* CHO cells transfection with LovTAP and RO to see if some expression can be detected
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* Culture of seed CHO cells (will be continued on 19th of July)
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{{:Team:EPF-Lausanne/Template/LabPresence|Sowmya, Alexandra, Mouna, June}}
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'''Transfection plan:''' different DNA percentages. Each one of those will have a duplicate.
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Made the Excel we use for our protocol, calculated total DNA with it. Here is the detail of the tubes we made:
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{{:Team:EPF-Lausanne/Template/Protocol|None}}
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Transfection Protocol -> CHO-DG44
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*Cell Density 3x10^6cells/mL
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*Total Volume 5 mL
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*DNA 1.5 µg/10^6cells
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*PEI 3 µg/10^6cells
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#Passage seed 1 day prior to transfection
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#Prepare tubes by addition of DNA
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#Centrifuge the necessary volume of seed, remove conditioned medium and resuspend in necessary volume of fresh medium to acheive the required cell density
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#Add 5 mL of the cell suspension to the tube with DNA and mix by orbitally shaking
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#Add the PEI to the Cell+DNA mixture as soon as possible
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#Place in the incubator at 37°C
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'''Transfection plan:''' different DNA percentages. Each one of those will have a duplicate.
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'''DNA concentration (Stock values) :'''
'''DNA concentration (Stock values) :'''
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'''Resuspension :'''
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'''Resuspension:'''
PCN found for 200µL of cell solution : 1.58
PCN found for 200µL of cell solution : 1.58
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We need a PCN of 0.2 for 1mio cells/mL -> 4mio cells/mL for 0.79 PCN.
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.
Therefore we needed to add 49mL of new medium.
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; Comments:
 
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We need to make a protocol page for CHO transfection
 
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{{:Team:EPF-Lausanne/Template/Footer}}
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Latest revision as of 23:42, 26 September 2012



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.

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