CALCULATING AMOUNTS OF NUCLEIC ACIDS

O.D. Units :
The "absorbance" of a chemical is a product of its (concentration) x (optical path length) x (extinction coefficient, E). Nucleic acids have a peak absorbance in the ultraviolet range at about 260 nm. When the spectrophotometer has a path length of 1 cm, absorbance = "optical density" (O.D.), and O.D. = E x concentration. Extinction coefficients vary with the type of nucleic acid. Double stranded DNA (dsDNA) has an E = 20 g-1cm-1L). Depending on the reference you read, the E for single stranded DNA (ssDNA) is 20 or 30 g-1cm-1L), while E for RNA is 25 g-1cm-1L).

Extinction coefficients can be used to estimate the concentration of a sam ple dissolved in a known aqueous volume, or to calculate the number of grams or moles of nucleic acid according to the following formulas:

Determining Concentrations:
      1 A260 O.D. Unit for dsDNA = 50 µg/ml
      1 A260 O.D. Unit for ssDNA = 33 or 50 µg/ml
      1 A260 O.D. Unit for RNA = 40 µg/ml

Examples:
A 1:50 dilution of dsDNA gives an A260 = 0.063. [DNA] = 0.063x50/20 = 0.16 mg/ml. This is for a 1 cm path length. The path length of the 5 µl cuvette is only 0.5 mm = 1/20 cm. Therefore multiplication by 20 to give a 1 cm path length and then dividing by 20 for the extinction coefficient cancel each other. So for that cuvette, [DNA] = (A260 - A320) x dilution factor. The A320 is used to subtract absorbance due to particles in suspension in this small chamber.


Determining Moles:
For ssDNA oligonucleotides, an estimate of the number of moles can be obtained by using an approximate mw of 350 Daltons/nucleotide. Accordingly,
1 O.D. Unit ssDNA = 33 x 10-6/(350 x length) moles = 94.3/length nmoles
An O.D. of 1.0 corresponds roughly to:
        10-mer        10 nmoles
        20-mer          5 nmoles
        50-mer          2 nmoles
      100-mer          1 nmole
Also, since 1 O.D. Unit = 33µg, then 1 µg = 2.86/length nmoles.
A precise estimate of the molecular weight of dephosphorylated oligos can be calculated using the formula:
      mw = [(#A's x 312.2)+(#G's x 328.2)+(#C's x 288.2)+(#T's x 302.2) - 61.0]1
For phosphorylated oligos:
      mw = [(#A's x 312.2)+(#G's x 328.2)+(#C's x 288.2)+(#T's x 302.2) + 17.0]1
For long (at least 30bp) dsDNA
approximate mw = 700 Daltons/base pair; mw of 1 kb is about 7x105 Daltons
      (649 Daltons/bp is more accurate, but harder to remember)
# µg in a pmole = length x 700 g/mole x 10-12 moles/pmole x 106 1;g/g
       = length x (7 x 10-4) µg/pmole

Multiply pmoles by 2 for pmoles 5' ends for dsDNA.
       ex: 1 kb DNA is about 0.7µg/pmole
      1 µg of a 1 kb DNA = 1.52 pmol; 3.03 (pmol ends)
      1 pmole of 1 kb DNA = 0.66 µg


MW RNA: length x 350 (341 is more accurate)

Other Useful Conversions for Oligonucleotides
      Oligonucleotide length to molecular weight
            350 x length = molecular weight
      Mass to µmoles
            µg / (350 x length) = µmoles
             (µg x 106) / (350 x length) = pmoles
      pMoles to µg
            pmoles x length x 350 /106 = µg
      µg to pmoles
           & nbsp;µg x 106/(length x 350)

       MW of oligonucleotide            pMoles 3' ends per microgram of DNA
              10 x 106            &nb sp;                               0.05
                1 x 106                         &nb sp;                  0.50
             0.1 x 106                                         & nbsp;  5.0
            0.01x 106                                           50.0

Melting Temperature Formulas (14-70 bases):
  &nb sp;   Tm = 81.5°C + 16.6(log[Na]) + 0.41(fraction G+C) - 600/L
      where
      Na = [monovalent cations] usually 50-60 mM
      L = primer length
      fraction G+C = G+C content of primer
      Also, Tm (approx) = 4°C x(fraction G+C) + 2°C x(fraction A+T)

Oligonucleotide Stock Concen trations for Amplifications:2
      mls to dissolve oligo = Total ODs (or A260) / (E x molar concentration needed)
      Count the numbers of each base in the oligo. Compute E
      = [#A's x (16,000)] + [#G's x (12,000)] + [#C's x (7,000)] + [#T's x (9,600)] + [#I's x (12,000)]
Make your oligonucleotide solution more concentrated than needed so that when you add it to a working solution, the final concentration will be correct.
      Ex:   18-mer with 2(A) + 6(G) + 8(C) + 2(T).
      E = 2(16,000) + 6(12,000) + 8(7,000) + 2(9,600) = 179,200
      Total ODs in tube = 11
      Concentrations needed = 5x10-4 = 0.5 mM
  &nb sp;   mls needed to dissolve = 11/(179,600 x 5x10-4) = 0.123 ml

Citations:
      1. Genosys Corp.
      2. Oligo, Etc catalog, p.14


    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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Copyright 1993, 1996, 1997, and 2001 by Mark Barton Frank, Ph.D. The author wishes to thank Andrea Masotti, Ph.D. Advanced Biotecnology Center - Centro di Biotecnologie Avanzate, Genova , Italy for clarification of an ealier versions of this text.
Proper citation for data acquired from this document is: "Frank, M. B. Nucleic Acid Conversion Factors: Moles - µg - O.D. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/dnamole.html). 1997. Oklahoma City. Revision Date: July 13, 2001."