what time is better to seed cells in well or dishes before transfection? - (Sep/12/2007 )
what time is better to seed cells in well or dishes before transfection?I mean how long will it take for the cells to be in a good condition after seeding them?does the time generallly almost fit most kinds of cell line?may someone help me,thank you.
this depends on your cells line, how you split your cells and how long you experiment will take after the transfection. Usually I plate them (HeLa, 293, Kyoto...) so that they will be 50-70% confluent the next day. At this stage I transfect them and 18-30h after transfection the protein is supposed to be expressed. However, it would be another protocol if you plate your cells for siRNA-transfection.
well i transfected hela cells and 293, roughly seeding plates at 19-20h for transfection at 10 the next day so appliying transfection mix at 11h)
it really depends on how fast the cells grow. The same cell line would grow at a different rate in different labs. Also you have to consider the passage numbers.
Had anyone tried to transfect hek293 immediately after seeding, as suggested in Fugene6 manual?
i transfect cells as soon as I split them, which means the cells are still in suspension form when I add the transfection mixture. They do pretty well. i use lipofectamine. But it should work for fugene as well.
I also perform lentivirus infection as soon as spliting the cells. Save time and effort.
Well it depends on many factors, ur cell line,their growth rate and the method of transfection you use and type of plasmid you are tranfecting, for lipofectamine 2000 for example cells should be 90/95% confluent to start transfection and as for the plasmid type if your plasmid is toxicto the cells or you are using triplet transfection its better for your cells to be 95% confluent.