Hi,
I will do transfection with fugene but, I tried to enter three plasmids in cell. There is no selection part of plasmids, and I will try to plasmids enter to in cell one by one. How to do this? Do you have any protocol for it?
I planned this protocol:
1) seed cell (1 million in 6 well), add pure dmem.
2) after one day of seeding, do transfection plasmid 1.
3) after one day of transfection, change media complete dmem.
4) after three day of change media, passage cells and seed complete dmem.
5) after one day of passage, change media with pure dmem.
6) after one day, do transfection with plasmid 2.
7) repeat 3-4-5 steps and do transfection plasmid 3.
Thank you...
You will need some form of screening that you are propagating cells that have been successfully transformed.
The protocol you are following is not apt. The chances of cells getting transfected by all three plasmids in this manner are very low. You should make a cocktail of all the three plasmids and then transfect cells with them. If you do not have a marker, you can run a qPCR for the target clones to see their expression in the cells.
@pankaj sharma I tried transfected to cells with 3 plasmids at the same time but it wasnot working. For that reason I am trying to find another solution 😔
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