Hi all, hope you will be able to help me. My protocol requires me to use 100-200 fmole amplicon DNA (let's assume I need 200 fmoles). The amplicon DNA concentration I have is 5.54 ng/ul and the size is 1200 bp. How to calculate how many microlitres I need to use to have the required 200 fmoles?
Thank you to anyone who could help me with that.
There are online calculators to do this - for example, see https://nebiocalculator.neb.com/#!/dsdnaamt. This is based on the following formulae:
moles dsDNA (mol) = mass of dsDNA (g)/((length of dsDNA (bp) x 617.96 g/mol/bp) + 36.04 g/mol)
moles of dsDNA ends = moles dsDNA (mol) x 2
DNA copy number = moles of dsDNA x 6.022e23 molecules/mol
You input the fragment length (1200 bp) and the amount of dsDNA (5.54 ng - we'll start with the amount of DNA in 1 µl). Assuming you've put the correct units and figures into the calculator you get 14.94 fmol (the concentration in 1 µl).
You want 200 fmol, so to get the volume you need you have to divide 200/14.94 = ~13.4 µl.
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