Calcium phosphate transfection question - (May/24/2006 )
Hi - I'm new to BioForum, but I did a search and didn't see an old thread on this (I may have missed one, though) - so please excuse me if this has been done to death in the past!
When doing a calcium phosphate transfection, what do you take as the zero time point? I'm doing a time series and can't decide when "0" would be - when I add the plasmid/CaPO4 mixture or when I remove that and add fresh media?
Thanks!
I would take the time when u add the plasmid/CaPo4 mixture to the cells as the start of transfection, as zero point.
hi
basically the protocol should say sthg like replace by fresh medium 3 hours post transfection.
So 0 is the time point when you add the mixture.
CaPO4 is a bit stressing for the cell, so depending on the experiment I would either consider time point 0 before doing transfection or as scolix and fred said after adding the plasmid, nevertheless, I wouldnt expect any significant expression or effect at that point other than the stress to the cell, correct me if am wrong
basically the protocol should say sthg like replace by fresh medium 3 hours post transfection.
So 0 is the time point when you add the mixture.
Thanks, everyone - I just wasn't sure how much plasmid the cells would take up right off the bat. I was mostly confused by the glycerol shock step, which is 4 hours after the plasmid/CaPO4 is added and would throw my time-course off a bit. But many protocols now skip the glycerol shock, so I'll stick with your ideas and take 0 as the initial plasmid hit. Thanks!