question about transfection - (Aug/31/2006 )
I am not sure whether anyone has already asked this question or not. Now I am doing transfection with fugene 6. In my case, for example, I transfect DNA A into both dishes, and then transfect DNA B into second dish but not first dish. To get same expression of protein A, we transfect empty vector(pcDNA3) into first dish at the same time for balance. My question is 'should we transfect same µg empty vector as DNA B or same mol empty vector as DNA B?' Also, another question :'normally, how many percent cells in second dish contain both DNAs?' Is that the more types of DNA transfected at the same time, the less percentage of cells containing all DNAs we will get?
Thanks in advance.
For your first question, you should use the same ug of the empty vector.
Are you plan to do two separate transfections? If you do not wait for long time, say less than one cell cylce, before the second transfection, and assuming you can achieve roughly the same transfection efficiency for both transfections, the % of transfected cells that have both DNA in theory would be roughly the (% of first transfection)^ 2. Of course many factors may affect the outcome of the experiment as you may already know.
Thanks, genehunter-1. I only do transfection once with two DNA into one dish, not seperately. Actually I tried using µg in my experiment, but the expressions of protein A in both conditions were not so equal, that is why I asked the question here. Where can I find some references which explains using µg instead of mol? I would like to know more clearly on this.
If you do co-transfection, you can adjust plasmid copy number the same using empty vector to fill in, such that total DNA applied is same for different plates.
If promoter/enhancer elements are same for both expression vectors, you would expect to see a close level of mRNA for both proteins. However, protein stability and metabolism rate may dictate the protein level.
I do not know if I understand your problem right; I remember co-transfections in our lab where co-overexpression of two proteins effected the expression of the other, i.e. protein A attenuated expression of protein B; so, if in co-expression assays, expression of one protein is lower than in control assays expressed alone, down-regulation by a co-expressed protein may happen & must be analyzed f.i. on the level of mRNA