Quantitation of DNA and RNA by Spectrophotometer
A spectrophotometer can be used to quantify the amount of nucleic acids in solution by measuring the absorbance at 260 nm (A260).
For the following nucleic acids, using a spectrophotometer (with a 10 mm path length) the A260 is equal to 1.0 for:
• 50 µg/mL double-stranded DNA (dsDNA)
• 33 µg/mL single-stranded DNA (ssDNA)
• 20–30 µg/mL oligonucleotides
• 40 µg/mL single-stranded RNA
The A260/A280 ratio is used as an indicator of nucleic acid purity. DNA has an A260/A280 ratio of ~1.8, and RNA has an A260/A280 ratio of ~2.0. Lower ratios can be caused by protein, phenol or guanidine isothiocyanate contamination. Absorbances of the main contaminants are:
• 230 nm: phenolate, thiocyanate and organic ions.
• 260 nm: protein contamination.
• 270 nm: phenol contamination.
• 280 nm: protein contamination, primarily due to the aromatic residues of proteins.
• 330 nm: particulates in the solution, which cause scattering of light in the visible range.
DNA Calculations for Ligations
MW of a double-stranded DNA molecule = (# of base pairs) X (660 daltons/base pair)
Moles of ends of double-stranded DNA molecule = 2 X (grams of DNA) / (MW in daltons)
Moles of ends generated by restriction endonuclease cleavage:
a) circular DNA molecule: 2 X (moles of DNA) X (number of sites)
b) linear DNA molecule: 2 X (moles of DNA) X (number of sites) + 2 X (moles of DNA)
Conversion of DNA: µg and pmol
µg DNA x pmol/660 pg x 106/1 µg x 1/N =pmol DNA where N= length of DNA in bp
• 1 µg of 1000 bp DNA = 1.52 pmol = 9.1 x 1011 molecules
• 1 µg of pUC18 or pUC19 DNA (2686 bp) = 0.57 pmol = 3.4 x 1011 molecules
• 1 µg of pBR322 DNA (4361 bp) = 0.35 pmol = 2.1 x 1011 molecules
• 1 µg of λ DNA (48502 bp) = 0.03 pmol = 1.8 x 1010 molecules