Bio-Rad SmartSpec Plus Spectrophotometer - Manual (Page 20)

Section 6
Operating Instructions
Note: It is Good Laboratory Practice (GLP) to use the SmartSpec Plus spectrophotometer
to measure absorbances in the range of 0.100 to 1.500 AU.
1. Choose an assay. SmartSpec Plus has six different assay keys in the second row of
buttons. When you press one of them, it signals SmartSpec Plus to clear its memory of
absorbance readings from the previous assay, to reset the sample number to 1, and to
reset the dilution factor to 1. Before the data buffer is cleared, SmartSpec Plus will
offer to print any unprinted data. All of the assays collect absorbance data and some of
the assays are designed to convert those readings into concentrations or other useful
information. The user interface of each assay is described in detail in the next section.
The available assays are:
A. DNA/RNA. In this assay, SmartSpec Plus will automatically collect absorbance
readings at 260 and 280 nm. If you choose to subtract the background at 320 nm
(in Setup), then the A320 reading is subtracted from the A260 and A280 readings
before the A260 and A280 absorbances are displayed or used in any calculations of
nucleic acid concentration or in calculation of the A260:A280 ratio.
Calculations. Nucleic acid concentrations of dsDNA, ssDNA and RNA are
determined by multiplying the A260 absorbance reading by a mass/absorbtion
conversion factor and by the dilution factor, or by multiplying the difference
between the A260 and A320 by the conversion and dilution factors.
Concentration = A260 * Conversion Factor * Dilution Factor
or
Concentration = (A260–A320) * Conversion Factor * Dilution Factor
SmartSpec Plus is programmed with default conversion factors for dsDNA,
ssDNA and RNA. These factors can be modified at any time.
Species A260 of 1.000
dsDNA 50.0 µg/ml
ssDNA 37.0 µg/ml
RNA 40.0 µg/ml
DNA and RNA oligonucleotide concentrations are reported in two different ways:
by mass (µg/ml) and moles (pmol/µl); you can toggle between the two values by
pressing the Conc key. In order to make these determinations, SmartSpec Plus
requires the molecular weight of the oligo and either the molar extinction coefficient
or the mass/absorbance conversion factor (as shown in the table above for dsDNA,
etc.). Frequently the molar extinction coefficient and/or the mass/absorbance
conversion factor is provided when you order an oligonucleotide from a custom
synthesis laboratory. However, if you don’t know this factor, SmartSpec Plus can
estimate them for you in one of three different ways. In order of increasing accura-
cy, by length of the oligo, by composition of the oligo, (i.e., how many A’s, C’s, G’s
and T/U’s) or by the primary sequence of the oligo. See Section 7.1 for a complete
explanation of the calculations (pg. 30).
B. Protein. The Protein function will automatically select the proper wavelength for
the Bradford (595 nm), Lowry (750 nm), BCA (562 nm) or UV Protein (260, 280,
and 320 nm) assays, or you can select a different wavelength for another assay.
The Bradford, Lowry and BCA are all well-characterized colorimetric assays for
protein concentration. The UV protein assay can determine protein concentration in a
nucleic acid background.
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