I am co-transfecting NIH3T3 cells with two plasmids (rasV12 mutant + gene of interest) for a transformation assay. My question is: how much reagent (FuGENE HD) should I use? I typically use a 3:1 reagent:DNA ratio for single transfections. But as I am adding twice the amount of DNA in total, should I use a 3:1 ratio for only one plasmid or both plasmids? Out of situations A and B below, which would you recommend?
Situation A: rasV12 (1 ug) + Gene X (1 ug) = 3 ul FuGENE HD
Situation B: rasV12 (1 ug) + Gene X (1 ug) = 6 ul FuGENE HD
B is the option to go for. However, you might try a 3.5:1 ratio for better results.
Good luck,
Best,
Hamadi
The ratio refers to TOTAL DNA. If you think this will cause the fugene volume to be too high you can try a 1:2 ratio, works for me in 293T cells. Just a tip, mix the plasmids before adding the media and fugene. It usually helps with homogeneity.
I agree with Luiza. I will go for 1 ug DNA in total (0.5 + 0.5) and 3 ul Liposome. 6 ul Liposome + 2 ug DNA in total (Option B) may be toxic to the cells. Regarding your aim, you may need to take the ratio of DNA X and DNA Y. For instance, one of your plasmids is the target and another one reporter (See the attached paper). You should use a higher amount of target DNA plasmid. Therefore, you can make sure all reporter carrying cells also express target gene. :-) However, if you need to co-express two genes equally you should go for the equal amount of plasmids. Good luck :-)
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