What factors should I consider when I optimize the efficacy of my calcium phosphate transient transfection?

You need to consider three main factors in order to optimize the efficacy of your calcium phosphate transient transfection. 

1. The amount of DNA in the calcium-phosphate-DNA co-precipitate – The total amount of DNA used in calcium phosphate transfection can vary widely among plasmid preparations and also with different cells and media. Most experiments use 10 μg – 50 μg total DNA in 450 μL sterile water. It’s advisable to test each new plasmid preparation and each new cell line being transferred for optimum DNA concentration. 
2. The length of time the cell is incubated with the co-precipitate – The optimal duration for incubating cells with co-precipitate varies with cell type. When using robust cell types such as NIH 3T3, HeLa or BALB/c 3T3, you will need to leave them in the co-precipitate for up to 16 hours for efficient transfection. However, more sensitive cells require shorter incubation times. 
3. The use and duration of glycerol or DMSO shock – Conducting a preliminary experiment varying the DNA amount, incubation time, and exposure to glycerol or DMSO shock can help you determine the tolerance of the cell type to prolonged exposure to a calcium phosphate precipitate and assess the need for glycerol shock. Analyzing the results of your pilot experiment will enable you to perform further optimization by further adjusting the experimental variables. For example, if your pilot experiment shows that transfection efficiency improves when cells are shocked with 10% glycerol for 3 minutes, subsequent experiments can explore variations in the duration of glycerol shock or the utilization of 10–20% DMSO shock.