Ok, so I'm having a little trouble understanding why I'm using the controls I've been using. Transfection is quite a new technique for me. The theory behind it is pretty simple, but it's performing it effectively that's got me in a bind. I feel it's because we're going a little overboard with the controls, and I'm not understanding all of the variables (I could be wrong).
So, what we've done is transfect HeLa cells with the pCR3 vector (Gateway cloning) and our gene of interest (GOI), infect the cells with a recombinant HSV-1-GFP, and then measure viral replication via the fluorescence of the recombinant HSV-1-GFP.
Here's what we have in our plate:
1. No cells transfected
2. Cells transfected with pCR3-GFP
3. Cells transfected with just the pCR3 backbone and no gene
4. Cells transfected with pCR3-ccdb (the destination vector)
5. Cells transfected with pCR3-GOI
6. Cells transfected with pCR3-mutant GOI
Each is performed in triplicate with and without viral infection. The results are normalized to 4 and 5 above when the background fluorescence (ie. non-infected cells) is subtracted from the infected cells.
What I don't understand is this: why use the purely the pCR3 backbone AND the destination vector, and why is it convenient to normalize to either of these? Why not just normalize to the non-transfected cells? Also, what's the point of transfecting with GFP? Is it just to show that the transfection worked?
The post-doc I work with is brilliant, but I think struggles to explain things concisely to me
Edited by greggers, 15 April 2013 - 01:43 PM.