Eppendorf BioSpectrometer kinetic - Manual (Page 93)

93
Evaluation procedure
Eppendorf BioSpectrometer ® kinetic
English (EN)
12.5 Special evaluation procedures for nucleic acids and protein UV
This section covers the evaluation of nucleic acids or proteins in the Nucleic acids and Proteins direct UV
method groups, as well as the corresponding biomolecular components in the Dye labels method group.
12.5.1 Correction A
260
and correction A
280
Application: correction of the influence of dye absorbance on the nucleic acid or protein absorbance at 260
and 280 nm for the methods of the
Dye labels group.
The application of the evaluation procedure can be activated in the parameters Correct A260 or Correct
A280.
A
XXX, corr
= calculated corrected absorbance for a wavelength of 260 nm or 280 nm
A
XXX
= measured absorbance for a wavelength of 260 nm or 280 nm
CF = correction factor for a wavelength of 260 nm or 280 nm (the correction factors for 260 nm and 280 nm
are both dye-specific and are programmed in
General Method Parameter: Dyes in the Functions area).
A
YYY
= measured absorbance at the dye wavelength.
12.5.2 Ratios A260/A280 and A260/A230
Application: Information on the purity of the measured nucleic acid. Application of the evaluation
procedure can be activated in the
A260/280 or A260/A230 parameters.
"Ratio" refers to the quotients of the measured absorbances at the listed wavelengths.
Literature values for ratio values with pure nucleic acids:
A260/A280
• DNA: 1.8 to 1.9
• RNA: 1.9 to 2.0
(Current Protocols in Molecular Biology, 1994)
A260/A230
For the ratios A260/A230, different information can be found in the literature for pure nucleic acids:
• DNA: 2.3 to 2.5
(The Nucleic Acids, 1955)
• DNA: 1.9
(Current Protocols in Molecular Biology, 1994)
The values are highly dependent on the pH value. Therefore, nucleic acids should not be measured in
water, but in a buffer with a pH of 7 to 7.2 (e.g., TE buffer).
The absorbance values displayed in the results are the directly measured, not the corrected
absorbance values.
YYY XXX corr XXX
A CF A A u
,