Dear all,
how microliter of DNA 1ug/ml I should apply in volume of dilution medium to get the final concentration is 1ug/ml in 96 well, 24 well and 6 well
Thank you for your help
Hello!
If you strictly follow the L2000 protocol, you should dilute your stock DNA at 1ug/ul in 250ul of medium (for 24-well). It means you have to add 5ug DNA to these 250ul, then it makes 5ul for you.
Then, you have a volume of 255ul of diluted DNA.
From this, you take 50ul (i.e. 1ug of DNA) and you mix it with 50ul of diluted L2000 (ratio 1:1).
You have now 100ul of L2000:DNA complexes (containing 1ug of DNA), enough to fill 2 wells with this plate format. The final amount of DNA per well is 500ng.
Hope it helps!
Last thing, I am not sure that L2000 is the best option for transfecting poly-IC. It's really designed for plasmid DNA. I would recommend to use a transfectant designed for both DNA and large RNA, like the Viromer RED from Lipocalyx. If you need more info, contact me at [email protected]
Bests,
Sandra
Hi Dyaningtyas,
I recommend optimize your protocol using different DNA concentration, that depend on your cell type, passages, etc; for a 6 well plate I use this, also make a working dilution of you DNA
For each transfection sample prepare transfection mixture: mix 5 ul lipofectamine to 100 ul Opti-MEM for 5 min, and meanwhile mix 2.5 ug DNA to 100 ul Opti-MEM; Combine these two mixtures and leave at room temperature (RT) for additional 5 min; Add the transfection mixture to 293FT cells and keep the plate in incubator for 48 h. For me is easier start the optimization in a 6 well plate. I use V1C1=V2C2 for my math. Hope it help
Vivian
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