2 replies to this topic

[soymilk14]

hi everyone. i just want to know what is the difference between recovery of cells transfected with plasmid(fused with gfp) for 12 hours, 16 hours and 24 hours? which would be more efficient to give a higher expression? The doubling time of my cells is 12 hours and i transfected 5 ug of plasmid in 5^6 cells.

thank you...

[cellgene]

soymilk14, on Apr 27 2010, 05:55 AM, said:

hi everyone. i just want to know what is the difference between recovery of cells transfected with plasmid(fused with gfp) for 12 hours, 16 hours and 24 hours? which would be more efficient to give a higher expression? The doubling time of my cells is 12 hours and i transfected 5 ug of plasmid in 5^6 cells.

thank you...

I do it at least for 24h, usually even for 48 hours so that you get more expression (but for 48h you should seed less cells of course) - and I do it both with 293T and HeLa cells.

[rkay447]

cellgene, on Apr 27 2010, 09:19 AM, said:

soymilk14, on Apr 27 2010, 05:55 AM, said:

hi everyone. i just want to know what is the difference between recovery of cells transfected with plasmid(fused with gfp) for 12 hours, 16 hours and 24 hours? which would be more efficient to give a higher expression? The doubling time of my cells is 12 hours and i transfected 5 ug of plasmid in 5^6 cells.

thank you...

I do it at least for 24h, usually even for 48 hours so that you get more expression (but for 48h you should seed less cells of course) - and I do it both with 293T and HeLa cells.

Unless of course, what you are going to express is toxic and kills the cells.  One of my constructs gives best expression and cell survival at 18-20 hours.  After that the cells die quickly and I'm left with the few cells that aren't expressing.  You may need to harvest samples at various times and run a western to determine when your specific protein is at the highest level.