For transfection we have used 500uL of transfection complex for 1 well of 6 well plate. so total media added to each well was 2mL. According to this 100nM of sirNA and 100ug/mL of nanoparticle was added in the transfection complex (500uL). What is the ratio of SiRNA to media. (we have a stock of 100micro molar of siRNA )
Also what if we lower down the volume of media to 50uL. Then how to maintain the ratio of siRNA to media?
Generally, siRNAs are transfected in the 50 - 200 nM range. You have a 100 nM solution of your siRNA in your media based on what you said. If you wanted to scale down the volume of your media (hopefully, you're not planning to do 50 µl per well in a 6-well), you need to scale down your transfection reaction in the same manner (i.e., 500 µl / (2000 µl / 50 µl) = 12.5 µl of transfection mix). You would only be using 0.05 µl of your 100 µM siRNA, which you would need to dilute (e.g., to 5 µM) before adding to pipette the proper volume accurately.
125ul optimem + 1,5ul 20microM RNAi
125ul optimem+2,5 ul RNAi max mix togheer and wait 20min RT
in the meantime replace the medium on your cells (i plate 2 10e5cells/2ml 6wells
with 1,25ml DMEM FBS witout ATbio
add the transfection medium overthe cells, mix..final volume 1,5ml analyse at 48 or 72Hours...work great (some people remove the medium after 2h and add complete media!)
work great see photo
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