Transfection problems - (Oct/07/2009 )
Recently I have been having problems carrying out transient transfection of 293T cells using Sigma Calcium Phosphate transfection kits. One day it works and the next day it does not under the exact same conditions. I have tried changing transfection kits, cells, and plasmid DNA. All the effort has failed to shed any light. There will be a day it works and the next it does not. After spending a whole of May trobleshooting this problem I finally got it to work but its stopped working again. I concluded that it must be the state of the cells because the plasmid DNA is fine and the transfection reagents are brand new(I have tried changing reagents). II plate out cells at 5X10^5 density/well on a six well plate.This gives me 50% confluence on the day of transfection. I found that below this and above this density my transfection efficiency goes down.I was wondering if anyone else has encountered similar problems? Any suggestion will be much appretiated.
I suggest going away from calcium phosphate, unless you have a particular reason for using it, to a lipid micell system such as Fugene or Lipofectamine - much more effective.
Tigz on Oct 7 2009, 01:56 AM said:
I agree with bob1, Lipofectamine and the other lipid trasfectants work great, particularly for 293 cells. I'll add that I have found, and it's Invitrogen's dirty little secret, that PEI works just as well as any of their reagents of this class (Lipo-2000, 293fectin, Freestyle MAX etc.), it is 1/100th the price from a chemical dealer and you use the exact same pocedure as the reagents I just mentioned.
I'm eagar to know how to prepare PEI reagents for transfection.
PEI= polyethylimine.
Totally agree, we also use PEI for plasmid transfections, it is easy and works perfect!
But I have never tried it for siRNA transfections and do not know if it works. I still use lipofectamine 2000 for that. Has anyone tried it?
wirly on Tue Nov 10 03:09:57 2009 said:
Recently I have been having problems carrying out transient transfection of 293T cells using Sigma Calcium Phosphate transfection kits. One day it works and the next day it does not under the exact same conditions. I have tried changing transfection kits, cells, and plasmid DNA. All the effort has failed to shed any light. There will be a day it works and the next it does not. After spending a whole of May trobleshooting this problem I finally got it to work but its stopped working again. I concluded that it must be the state of the cells because the plasmid DNA is fine and the transfection reagents are brand new(I have tried changing reagents). II plate out cells at 5X10^5 density/well on a six well plate.This gives me 50% confluence on the day of transfection. I found that below this and above this density my transfection efficiency goes down.I was wondering if anyone else has encountered similar problems? Any suggestion will be much appretiated.
I agree with bob1, Lipofectamine and the other lipid trasfectants work great, particularly for 293 cells. I'll add that I have found, and it's Invitrogen's dirty little secret, that PEI works just as well as any of their reagents of this class (Lipo-2000, 293fectin, Freestyle MAX etc.), it is 1/100th the price from a chemical dealer and you use the exact same pocedure as the reagents I just mentioned.