Hi,
I am trying to perform a luciferase assay using Applied Biological Materials (abm) plasmids, but I am uncertain on how much plasmid DNA should I use. I already decided that I will plate the cells in 24-well plates, and I know the amount of mimic miR that I will co-transfect the plasmids with but I can't figure what is the best plasmid DNA (WT 3'UTR, mut 3'UTR and empty vector) quantity, per well, to use.
I've been reading (a lot) and most authors fail to mention the DNA quantity used and when they mention, there isn't a uniform quantity. I've already read reports in which authors used 50 ng/well while in others, used 1 ug/well.
If anyone already used this plasmids and knows how to proceed, I would be extremely grateful if you could help me.
Best regards,
Gonçalo Pinho
It depends on the plate you are using (96, 24, 12, 6)wells and the transfection reagent. So what you need is to start optimizing your experiment based on the transfection reagent you are using, e.g. for 96 well plate starting with 50ng, 100ng, 150ng to check which concentration is the best. I am transfecting luciferase plasmid (50ng) using jetprime in 96 well plate and worked perfectly. So you can find the recommended conc. to be used on the protocol of the transfection reagent.
For 24 well plate,jetprime transfection recommended 0.25 - 0.75u of DNA.
Hi Goncalo,
I have never used these specific plasmids you are describing before. But I agree with Ammar that you should first do an exploratory experiment to see which concentrations work best for you, starting with the suggested ranges from the datasheet. Personally, when I have done plasmid transfection of mammalian cells before - to express a protein of interest - I have used 1 to 2 μg of plasmidDNA in each well of a 6well plate.
When I need to use different sizes of plates (e.g. 24well plate), I calculate the final DNA to use by doing the proportion, in relation to the surface area of the wells. Which in this case would work out ~ 0.2 - to 0.4 μg of plasmid. However, I think is always best to test at least three concentrations and if you can, try to estimate the transfection efficiency (how many cells have been successfully transfected), especially if you are not planning to do antibiotic selection; so that you can have a good idea of how many cells are actually going to be expressing your constructs in each well. Do these plasmids contain GFP/RFP markers?
Good luck with your experiments!
Hi Colleague
The optimal ratio of transfection reagent to DNA is highly cell type-dependent. As a starting point, the amount of transfection reagent should be varied while keeping a constant plasmid DNA concentration (for example, 1:1, 3:1 and 5:1 ratios of volume to mass).
Visit the following url ay help you:
https://www.thermofisher.com/iq/en/home/references/gibco-cell-culture-basics/transfection-basics/guidelines-for-plasmid-dna-transfection/optimizing-plasmid-dna-transfection.html
Regards
Ammar Mohammed Faiz Yousuf Ahmedani, thank you so much for your answer. Since I am going to use 24-well plates, I will try the plasmid DNA range that you suggested (0.25-0.75ug/well). Regarding the transfection reagent, I will not use the one you mentioned, and instead will use Lipo3000, which I believe will work perfectly.
Once again, thank you for your help.
Clara Apicella, thank you for your answer. Due to Luciferase's protocol, I won't be able to perform positive-clone selection.
Since the amount of plasmid DNA that I have is limited, even tho I will first amplify it, the range that you suggested is quite interesting, and if it works, might greatly reduce the amount that I need, so thank you very much for the suggestion.
Lastly, my plasmids do not have GFP/RFP markers. I decided to design them with the Luciferase dual system (Luc and Ren), instead of GFP or other tag.
Prof. Ayad Ghany Ismaeel, thank you for your answer. I will certantly look into the link that you sent.
Hi Gonçalo Outeiro-Pinho
In the end it probably doesn`t matter in your case to know the proportion of the cells that are transfected, since you will measure luciferase activity in control wells and reduced luciferase activity in miR transfected wells, I imagine. When testing the concentrations, I would just do it in triplicates so you can have an idea of the average luciferase activity you can measure with each concentration. Maybe you can try 0.2, 0.4 and 0.6 ug to start with. If you are not happy with the results, you might want to adjust the amount of transfection reagent you use (e.g. Lipofectamine...).
Good luck! I hope your experiments work out well!
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