Help! Ca Phos transfection not working - transfection (Apr/03/2006 )
Hi I was wondering if anyone would be able to help. So here it is:
Transfecting using Ca phopshate ppt.
The cell lines which we have used in the lab for years suddenly stop transfecting.
The cells are mycoplasma FREE, bought in new cells line just in case and they wont transfect!
The pH of HBS was tested and is fine, even bought in HBS, CaCl2 also bought in and no difference.
The cells look healthy, and are growing well.
Plasmid preps for control are the same as used before.
Media and antibiotics the same.
Even transfection with lipids doesn't work.
Any ideas?
Please Help,
thank you
how do you test transfection efficiency?
Are you constructs GFP fluorescent?
I am trying to find out if indeed your transfection is not working or the detection method is not working.
Are you constructs GFP fluorescent?
I am trying to find out if indeed your transfection is not working or the detection method is not working.
Its a luminescence measurement pGL3 control, substrate seems ok.
GFP also very low in transfected cells
What is the passage number of your cell line? Can it be too old?
Maybe you could try thawing a new vial from your stock and culture it.
Maybe you could try thawing a new vial from your stock and culture it.
Hi,
We bought in new cell lines (really that desperate!) still nothing, last week.
Really strange eh?
Does ur luminometer work?? You might want to make sure of that. Try to see if you can read your samples in another luminometer and compare the reading. Use the same substrate though...
Another reason that comes to mind is the PGL3. Did you buy new plasmid or did you make it yourself. If it is the latter then and you have used the JM100 series of bacteria to make the plasmid you might be having an endotoxin problem.
Hang in there...it will workout...Let me know how u do it exactly. I had similar problems in the past. My guess is it might be the plasmid or ur luminometer since 1. ur cells are new 2. CaPhos nor lipid seems to work..
endotoxins you would see by you're cells dying or detaching. (I've done transfections with GFP-reporter on sensitive cells with DNA that wasn't endofree and cells were EGFP+ but floating around).
Apart from that: do check your luminometer!
Apart from that: do check your luminometer!
Thank you, we had the same idea and have dug out the original pGL3 plasmid to check if its the new plasmid preps, hope it works. Will let you know.
Thanks again