uM molecule versus basepair calculation
Posted 14 December 2010 - 05:19 PM
Scenerio 1) Whenever purchasing primers, they usually come lyophilized in nmol that is calculated from A260 and converted with the epsilon value to get a nmol in molecule form, not nmoles of basepair so length matters. ie: 107 nmols = 107 nmols of molecules, not 107 nmols of bases.
Scenerio 2) When i convert dsDNA from nanodrop ng/ul, i usually convert by 660 g / 1 mol bp without taking into account the dsDNA length, giving a uM in bp rather than molecule. Units can still come out to uM meaning micromoles of basepair / L.
My question: Is it general practice to assume moles in DNA conversions automatically refer to moles of molecules rather than moles of basepair, and as such I should add in the length of the DNA in the calculation.
Example online calculators such as promega and ABI / roche take into account the length or molecular weight (which indirectly takes into account the length) and label the answer as pM for example, but don't specify pmoles of molecules / L or pmoles of basepairs / L. The calculations do correspond to pmoles of molecules / L but I wanted to see whether this is general practice throughout the community. Nanodrops give a tangible ng/ul value taking into account the physical bases via beers law does it not?
Thanks and hope it's not too confusing.
Posted 15 December 2010 - 11:10 AM
genius does what it must
i do what i get paid to do
Posted 17 December 2010 - 08:31 PM
Edited by fume711, 17 December 2010 - 08:32 PM.
Posted 18 December 2010 - 07:52 PM
Unfortunatly you have been calculating your concentrations incorrectly - though they are easily converted if you know the length of your DNA/RNA.