Transfection Problem - (Dec/25/2010 )

So I have been having trouble with a transfection of a particular DNA
Here is what I think may be one possibility but I do not know if this can happen, and that is where I need the help...

The cells that I am using are 293T but they were passed down from the last person to have this project... often the cells were not cared for until the media (DMEM) was completely yellow and the cells were hanging on by a thread. Those cells had been passed about 5 times by this person and frozen down- often the freezing process was no good either because they did not keep the cryovials on ice or take them immediately to the -80 after adding the DMSO which is highly toxic. So there is an overview- now I am using these cells and I have been thawing their stock and growing up what I think are healthy cells but the problem is he has not had a successful transfection with this DNA for about six months- we know that this DNA is very difficult and it also is very hard to clone, and such- my question is- can the cells somehow incorporate some type of mutation in themselves (because of their un cared for living conditions) that decreases their transfection efficiency? And the only reason the positive control is working is because it is FADD? Because the problem is- this DNA was transfecting perfectly, and then out of no where stopped working- I was wondering if this is because of the cells, because the construct is great and the DNA prep that had been working when recently checked, did not work- which leads me to think it could only be the cells at this point.

I would GREATLY appreciate ANTYHING!! I have been troubleshooting this for about 2 months with no success and it is very frustrating for myself and my PI.

Thanks!
FC

if you think cells are a problem, but new 293's from ATCC. you are sure about the quality of DNA?

The DNA has been sequences MULTIPLE times and it is in frame (your other reply)... We have checked all of these- and I have no idea what else there is that I can troubleshoot- even when a recent Transfection was done with the DNA that had been working it didn't this time... That is why the only thing I can think of is the cells- I have recently thawed a vial that was from the original stock, so hopefully it works- but I wanted to know if this was possible, that there be something that happens to the cells from not being well cared for? That would cause them to just stop transfecting a particular DNA? It just seems crazy to me.

If cells have been passaged for long, they change a lot of properties including transfection ability but thats for all plasmids not for one particular plasmid.

good luck