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  • FAQ: How to calculate the molarity of ends?

    NEB suggests using the free online tool, NEBioCalculator. This tool will calculate molarity of ends as well as other biomath calculations.

    If you would like to calculate “by hand”, please note the following:

    Molarity = [(µg/µl) ÷ (base pairs x 650 daltons)] x 2 ends

    Example:
    • Calculate the molarity of ends for a linearized 5 kb vector that has a concentration of 250 ng/µl: [(0.25 µg/µl) ÷ (5000 x 650 daltons)] x 2 = 154 nM
    • Calculate the molarity of ends if you put 50 ng of this vector in a 20 µl ligation reaction:
      50 ng ÷ 20 µl = 0.0025 µg/µl 
      [(0.0025 µg/µl) ÷ (5000 x 650)] x 2 = 1.54 nM
    • Determine the amount of a 1 kb insert needed to achieve a 3:1 insert:vector ratio:
      3 X 1.54 nM = 4.62 nM 
      [(X µg/µl) ÷ (1000 x 650)] x 2 = 4.62 nM 
      0.0015 µg/µl x 20 µl = 0.03 µg = 30 ng
    • Does this reaction fall within the optimal range of 1–10 µg/ml?
      (50 ng vector + 30 ng insert) ÷ 20 µl = 4 µg/ml