too few cells used for electroporation/ transfection - (Dec/18/2008 )
hi, i just did my first transfection and to my horror, realised that i calculated the cells wrongly such that i only used 2/3 the number of cells required. (0.6million instead of 10million). I only realised this the day after when i checked the cells and saw that the cells are really sparse (looks like 30% confluency a day after electroporation). My question now is does anyone know how is it going to affect my subsequent luciferase result? Will using more cell lysate for luciferase reading help? Do you think the electroporation will be affected such that the transfection efficiency will be much lower than usual due to the low cell count?
Just to make things clearer, i'm supposed to use 10million cells for electroporation.
Thanks in advance.
Regards,
I see no chance to get reliable results only by adding more lysate.
But: Don't you have any possibility for normalisation? We use the DualGlo-System and have therefore an internal standard to normalize for different cell numbers. Or without this system I did at least a few control transfection to normalize for.
I never thought about comparing different luciferase experiments without any standard. How do you know if you always have the same transfection efficiency? Or if your cells divide exactly the same in each experiment?
this might sound a bit simple but can you not wait until your cell number has insreased to what you would normally use?, i.e 90 % confluent or whatever confluency you normally use?
if the transfection has worked then back calculate using 0.6 instead of 1, i think the only error you really made is that there might not have been enough cells for the transfection to work but i pressume 600,000 cells is plently for any transfection
if the transfection has worked then back calculate using 0.6 instead of 1, i think the only error you really made is that there might not have been enough cells for the transfection to work but i pressume 600,000 cells is plently for any transfection
No, you can't. There's a difference between transient and stable expression. Even with stable clones the ratio of transfected and untransfected cells would be different to the ratio after infecting more cells in the beginning.
Just letting them grow to the desired confluency is a bad thing to do, for one, you have no measure of how many cells are actually there, meaning you can't easily compare between experiments. Another problem is that you get loss of transient expression and different growth rates depending on which part of the (and which type of) plate your cells are seeded in.