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Libra S22, English 2
This section is only a description of the additional Life Science modes now
included with every Libra S22 spectrophotometer.
For a complete description of all other modes and the general use of the
spectrophotometer, please refer to the main manual included on the CD-ROM.
Life Science mode includes following applications:
•Stored parameters for Nucleic Acid quantification and purity checking
-DNA
-RNA
-Oligonucleotide
Nucleic Modes (3)
Nucleic acids can be quantified at 260 nm because it is well established that a
solution of DNA or RNA with an optical density of 1.0 has a concentration of 50 or
40 µg/ml, respectively, in a 10mm pathlength cell. Oligonucleotides, as a rule of
thumb, have a corresponding factor of 33 µg/ml, although this does vary with base
composition.
Extracting nucleic acids from cells is accompanied by protein, and extensive
purification is required to separate the protein impurity. The 260/280 ratio gives an
indication of purity; it is only this, however, and not a definitive assessment. Pure
DNA and RNA preparations have expected ratios of ≥1.8 and ≥2.0, respectively;
deviations from this indicate the presence of protein impurity in the sample, but care
must be taken in interpretation of results. An elevated absorbance at 230 nm can
indicate the presence of impurities as well; 230 nm is near the absorbance maximum
of peptide bonds and also indicates buffer contamination since Tris, EDTA and other
buffer salts absorb at this wavelength. When measuring RNA samples, the 260/230
ratio should be > 2.0; a ratio lower than this is generally indicative of contamination
with Guanidinium Thiocyanate, a reagent commonly used in RNA purification and
which absorbs over the 230 - 260 nm range.
Background correction at a wavelength totally separate from the nucleic acid and
protein peaks at 260 and 280 nm, respectively, is sometimes used to compensate for
the effects of background absorbance. The wavelength used is 320 nm and it can
allow for the effects of turbidity, high absorbance buffer solution and the use of
reduced aperture cells.
