I would usually first look at the manufacturers suggestion, and then choose a few different volumes higher and lower to determine empirically the best combination of lipid to oligo, and check knockdown efficiency by Western blot or qPCR. This will vary between cell types, and between different oligos. Some combinations are more toxic to the cells than others, so you need to check this too. From memory, Lipofectamine in HeLa cells in a 6 well dish (i.e. 2ml volume) was 5 ul of lipid to 6.25 ul oligo (from a 20 uM stock) - would be a good starting point to test from
Hey Rob thanks a lot.. I have standardised amount of lipo in case of plasmid transfections. Generally DNA:lipo ratio of 1:2 works fine. So accordingly if i reduce the concn of siRNA from 100nM to 50nM, shud i reduce the amount of lipo. or does it depend only on amount of medium used for transfection ?
In my experience, both the ration of lipofectamine to oligo and the total volume used affect the efficiency
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