How to Calculate a DNA Primer Concentration
Calculations can be the bane of laboratory work. Therefore, I have always looked for easy methods for getting the math done. Here are several different ways to calculate primer concentration depending on the starting material. For all calculations, let’s assume we have 22 nmol of a DNA primer containing 16 bases.
From lyophilized powder
Primers purchased from companies will often be sent as a lyophilized powder, and the company will tell you the nmoles of primer they have provided. If you are confident that you have recovered all of the powder and have had no previous issues with the amount of primer sent, then a quick resuspension and calculation can be done without measuring the OD of the primer. If your downstream application with the primer is highly sensitive to primer concentration, or you are unsure of the quality of primer, you should measure the OD of the primer after resuspension and use the second method for calculation.
1) Resuspend the primer in 100 ul of water or buffer and use the following calculation:
(XX nmol/100 ul) x (1000 pmol/nmol)= pmol/ul = uM
Example: (22 nmol/100 ul) x (1000 pmol/nmol) = 220 pmol/ul = 220 uM
2) Alternatively, you can quickly resuspend a primer at 100 uM by resuspending the powder in a volume in microliters that is 10x the number of nmol.
Example: Resuspend the 22nmol of primer in 220 ul=100 uM
From a liquid
Sometimes I have not always been the good scientist that I mean to be and find tubes of primers without concentrations written on them. Or I have occasionally found that I do not recover the full amount of primer sent lyophilized from a company. In these cases, primer concentration is easily calculated from an OD260 reading. Let’s assume we diluted the primer from above 1:200 and the OD260 reading was 0.132.
The concentration can be calculated using the following formulas:
(OD260) x (0.02*) x (dilution factor) = ug/ul *Conversion factor for single stranded DNA
(ug/ul/330Daltons/nt**) x (106/# of nt) = uM **Average MW per nucleotide
Example: (0.132)(0.02)(200)=0.528 ug/ul
(0.528/330) x (106/16)= 100 uM