I'm performing transfection on HeLa cells using 24-well plate in triplicate. I discovered that after I harvested the cells, alot of cells lost when I removed the supernatant after spinning down the cells. Sometimes, I even get 0.4mg/mL of measured total protein which is impossible to be loaded into minigel to get 20ug of protein per lane. Can I pool all the cells from triplicate into one tube (Is this still reliable in terms of doing replicate in experiment)? Is there any technique I should consider when removing supernatant after spinning down the cell? Generally I just pour away the supernatant, absorbed the residue on tissue, but I use pipette to remove the medium which was still left at the tube bottom due to surface tension.
1 reply to this topic
Posted 16 December 2009 - 09:08 AM
You can pool your samples, but you won't be able to consider the experiment done in triplicate (at least not properly done) since you'll have no error bars from whatever assay you're running on the cells. You can try removing the sup slowly by pipette instead of pouring. Or, what is your total volume of 0.4mg/ml - maybe you can just lyse in a smaller volume?