The plasmid molarity required for my transfection is 2.75µM.
I don't know the initial molarity of my plasmid, I know just the lyophilized micrograms (5 µg) I have now in my tube and the kb of my plasmid (7.5kb).
Before to reconstitute it I would like to understand how can I calculate the initial molarity.
Could please someone help me?
You can calculate the molecular weight of the plasmid with the average molecular weight of monophosphate deoxynucleotide of 330. So for a 7.5kb plasmid, the mw is 4.95e6. I have never heard of using a molar concentration of DNA in transfection protocol as each DNA would have a different size. You should double check your protocol.
Federica Scalia. You can input your plasmid sequence to the following online resource https://www.bioinformatics.org/sms2/dna_mw.html with double-stranded and circular selected for the parameters to obtain the molecular weight (in Daltons which is equivalent to g/mol). Then simply insert this value for molecular weight, 5micrograms as the mass and the volume (will be the volume of water you plan to resuspend the plasmid in) in the formula for molarity. Since you mentioned initial molarity, molarity comes into effect only in a solution so water ever volume you plan to resuspend now will determine its molarity at this point. Hope this helps.
Bill Chi Shun Ho Thank you!
I did these calculations and "unrealistic" numbers for a transfection come out.
Also for me it is the first time that I read molarity instead of nanograms. I am using Viromer blue for the first time, I have checked several times and it requires 2.75uM of the plasmid. It is very strange.
Akhil Baby Thank you!
I tried to do these calculations taking in consideration to resuspend my plasmid in 50ul, but the number is kind of weird.
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