I will be using Lipofectamine 3000 for plasmid transfection in A549 cells. I plan to test different concentration of plasmid that can give best result. so i decided to test 1ug, 2.5ug and 5ug of plasmid. i will be performing transfection in 24-well plate. But as in protocol, each 1ug DNA, 2ul of the p3000 reagent is required. Then the volume of diluted DNA is no longer 1:1 ratio with the volume of diluted optimem. Can i just adjust the optimem volume so that the ratio will be 1:1? after i add both diluted dna and diluted optimem, the volume of mixture already 75ul which is exceed 50ul that is recommended by protocol. Can someone help me how to adjust the volume? is it ok if i just use the volume that i get
Hi,
you should dilute DNA and P3000 in a total of 25uL, each.
Then, you combine the two tubes (25uL) for a total of 50uL. This gives a ratio of 1:1, volume/volume.
What is the concentration of your plasmid? If you have to go beyond 25uL, its most likely very low... you could concentrate it to achieved a working solution of 0.5-1ug/uL.
In attachment, I've include the calculations to show the 1:1 ratio (volume/volume).
Best,
Nicolas
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