Bio-Rad SmartSpec Plus Spectrophotometer - Manual (Page 33)

• Press the right arrow to continue the assay. SmartSpec Plus will now be ready
to measure the absorbance of your oligonucleotide and to convert those
absorbance data into concentrations of µg/ml and pmol/µl.
Nucleic Acid Calculations
Molecular Weight (MW):
• Length (L): MW ≈ L * 310 - 61
• Sequence or Composition:
DNA: MW = (#A * 312.2) + (#C * 288.2) + (#G * 328.2) + (#T * 303.2) - 61
RNA: MW = (#A * 312.2) + (#C * 288.2) + (#G * 328.2) + (#U * 289.2) - 61
Molar Extinction Coefficient ( εε):
• Length: ε ≈ L * 10,000 liter/mole/cm
• Composition:
DNA: ε ≈ (#A * 15480) + (#C * 7340) + (#G * 11760) + (#T * 8850) liter/mole/cm
RNA: ε ≈ (#A * 15480) + (#C * 7340) + (#G * 11760) + (#U * 9000) liter/mole/cm
• Sequence: See Optical properties of nucleic acids, absorption, and circular dichroism
spectra, p. 589, CRC Handbook of Biochemistry and Molecular Biology 3rd
Edition, Nucleic Acids - Volume I. Gerald D. Fasman, Ed. CRC Press 1975.
III. DNA and RNA Oligonucleotide Concentration:
• Concentration = A/( εb) * 10 6 pmole/µl
• Concentration = A/( εb) * MW * 10 3 µg/ml
where A = absorbance, ε = molar extinction coefficient in liter/mole/cm,
b = pathlength in cm and MW = molecular weight.
For cuvettes with a 10 mm (1 cm ) pathlength, these equations simplify to:
•
Concentration = A/ ε * 10 6 pmole/µl = (µg/ml)* (1000)/MW
• Concentration = A/ ε * MW * 10 3 µg/ml = (pmol/µl) * (MW)/1000
IV. dsDNA, RNA, ssDNA concentration:
• A
260
* Conversion Factor * Dilution Factor = µg/ml concentration
OR
• (A
260
- A
320
) * Conversion Factor * Dilution Factor = µg/ml concentration
Default conversion factors are:
1.0 O.D. unit = 50 µg/ml dsDNA concentration
1.0 O.D. unit = 40 µg/ml RNA concentration
1.0 O.D. unit = 37 µg/ml ssDNA concentration
A
260
/A
280
ratios are 1.8 and 2.0 for pure DNA and RNA preparations, respectively.
30
Mol ext coef= __________
MW= _________ >=Continue