Eppendorf D30 BioPhotometer - Manual (Page 77)

77
Evaluation procedure
Eppendorf BioPhotometer ® D30
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.
12.5.1 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).
12.5.2 Conversion to molar concentrations and nucleic acid quantities
The conversion only can be applied to nucleic acids. It is realized in the process results/More
calculationsmethod step.
12.5.2.1 Calculation of amount
Application: calculating the amount (mass) of nucleic acid in the total sample volume.
M = calculated total amount (mass) of nucleic acid in the sample tube. Unit: μg.
C = nucleic acid concentration calculated from the measurement. Unit: μg/mL or ng/μL.
V
S, total
= total volume of the sample in the sample tube. Enter this value in More calculations. Unit: μL.