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| | <!-- Don't forget to add this protocol to the protocol list page: --> | | <!-- Don't forget to add this protocol to the protocol list page: --> |
| | <!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol --> | | <!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol --> |
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| | + | 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 [https://static.igem.org/mediawiki/2012/4/49/Team-EPF-Lausanne_CHO_Transfection_excel_protocol.xls 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. |
| | + | |
| | | | |
| - | *Cell Density 3x10^6cells/mL
| + | 1. Passage seed 1 day prior to transfection. |
| - | *Total Volume 5 mL
| + | |
| - | *DNA 1.5 µg/10^6cells
| + | 2. Prepare tubes (yellow caps with holes) by addition of the calculated amount of DNA. |
| - | *PEI 3 µg/10^6cells
| + | |
| - |
| + | 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). |
| - |
| + | |
| - | #Passage seed 1 day prior to transfection
| + | 4. Add 5 mL of the cell suspension to the tube with DNA and mix orbitally. |
| - | #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
| + | 5. Add the PEI (45 µl) to the Cell+DNA mixture as soon as possible, flick 3 times. |
| - |
| + | |
| - | #Add 5 mL of the cell suspension to the tube with DNA and mix by orbitally shaking
| + | 6. Place in the incubator at 37°C. |
| - | #Add the PEI to the Cell+DNA mixture as soon as possible
| + | |
| - | #Place in the incubator at 37°C
| + | |
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| | {{:Team:EPF-Lausanne/Template/ProtocolFooter}} | | {{:Team:EPF-Lausanne/Template/ProtocolFooter}} |
| | <noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude> | | <noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude> |
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