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LabelGuardTM Mikroliter Messzelle The NanoPhotometerTM

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The NanoPhotometerTM is equipped with pre-programmed methods for highest user comfort. Nucleic acids - dsDNA, ssDNA, RNA, and Oligonucleotides Labeling efficiency - dye incorporation rate for microarray experiments Proteins - Bradford, Lowry, BCA, Biuret, and UV Protein (A280) Cell density - for microbiology and cell cultures Nucleic Acids Application Nucleic Acids Quantification It is well established that for determination nucleic acid concentration in solution the absorbance at wavelength 260 nm (A260) is used. The function describing the concentration to absorbance relation is the Lambert-Beer Law: A = e * c * d. The absorbance (A) is the product of the substance specific extinction coefficient (e), the concentration of the absorbing sample (c), and the optical pathlength in cm (d). A solution of dsDNA in a 10 mm pathlength cell with an optical density of 1.0 has a concentration of 50 µg/ml. Therefore, nucleic acids can be quantified at 260 nm using for different type. The NanoPhotometerTM uses factors 50, 40, 37 and 33 as defaults for dsDNA, ssDNA, RNA and oligonucleotides, respectively, and compensate factors for dilution and varying pathlength (10 mm, 5mm, 2 mm, 1 mm, 0.1 mm). Nucleic Acids Estimation of Purity Depending on the extraction/purification or synthesis/purification method of the nucleic acids different impurities can be expected (TRIzol, humic acids, carbohydrates, Guanidine thiocyanate, nucleotides, peptides, EDTA, phenol and protein). It is recommended to include OD ratio measurement (A260/A280 and A260/A230) for purity estimation. Pure DNA and RNA preparations have expected ratios of ³ 1.8 and ³ 2.0, respectively; deviations from this indicate the presence of impurity in the sample, but care must be taken in interpretation of results. Ratio A260/A280 In solution, pure DNA and RNA typically have A260/A280 ratios of 1.8 and 2.0. If the absorbance ratio is significantly less, the nucleic acid is probably contaminated with protein. Accurate quantification of nucleic acid is not reliable without prior purification, and the efficacy of this can be judged by the A260 /A280 ratio. Ratio A260/A230 For RNA samples the ratio values <2.0 point out genomic DNA contamination. Successful DNase I treatment displays in ratio values >2.0. Ratio values <1.5 indicate impurities of extraction chemicals or incompletely removed constituents of cells. Note: Both ratio values can also be perturbed easily by pH, even if the nucleic acid samples are clean. Use buffers around 7.5 for your measurements. The user can switch between two option of result presentation: a data table or a scan plot. Use of Background Correction Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280nm, 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. The NanoPhotometerTM offers the option of background correction at 320 nm for nucleic acid determination. Dye incorporation rate To determine the dye incorporation rate, the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used. The corresponding extinction coefficient of the dye is used in the Lambert-Beer Law to determine the dye concentration (c = A / (e * d)). Comparing these values with the DNA concentration gives a dye incorporation rate. FOI = C(dye) / C(nucleic acid) Example: Frequency of Incorporation (FOI) of Cy3 per 1000 bases: FOI(Cy3) = 58.5 * A550/A260 The user can switch between two options of result presentation: a data table or a scan plot. Use of Background Correction The absorbance reading of dye labelled nucleic acids at 260 nm is affected by the dye contribution. To obtain accurate concentration values, the contribution has to be eliminated using correction factors. The NanoPhotometerTM offers the option to select the background correction for dye contribution of absorbance reading at 260 nm for nucleic acid determination. Protein Applications 1. Protein UV

The Protein UV method determines Proteins at 280nm. The effect of nucleic acid in the protein solution due to strong nucleotide absorbance at 280 nm can be compensated by measuring Abs 260, and following the equation of Christian and Warburg: Protein (mg/ml) = 1.55*A280 - 0.76* A260 This equation can be applied to other proteins with known corresponding factors or determinate at known protein concentrations. The use of background correction at 320 nm is optional.

2. BCA

The BCA method depends on reaction between Cupric ions and peptide bonds, but in addition combines this reaction with the detection of Cuprous ions using bicinchoninic acid (BCA), giving an absorbance maximum at 562 nm. The BCA process is less sensitive to the presence of detergents used to break down cell walls. Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).

The user can switch between two options of result presentation: a data table or a scan plot.

3. Bradford

The Bradford method depends on quantitating the binding of a dye, Coomassie Brilliant Blue, to an unknown protein and comparing this binding to that of different, known concentrations of a standard protein at 595 nm (default setting); this is usually BSA, bovine serum albumin. Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).

The user can switch between two options of result presentation: a data table or a scan plot.

4. Lowry

The Lowry method depends on quantifying the colour obtained from the reaction of Folin-Ciocalteu phenol reagent with the tylsryl residues of an unknown protein and comparing with those derived from a standard curve of a standard protein at 750nm; this is usually BSA, bovine serum albumin Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).

The user can switch between two options of result presentation: a data table or a scan plot.

5. Biuret

The Biuret method measures the reaction between Cupric ions and peptide bonds in an alkali solution, resulting in the formation of a complex absorbing at 546 nm. Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).

The user can switch between two options of result presentation: a data table or a scan plot.

OD 600 – Cell Density Applications

The method OD 600 is used to determinate the absorbance at 600 nm. Bacterial cell cultures are routinely grown until the absorbance at 600 nm (known as OD600; default setting) reaches approximately 0.4 prior to induction or harvesting. A linear relationship exists between cell number (density) and OD 600 up to approx. 0.6. Select units of measurement: OD, cells/ml. With units OD selected the results output are OD values. If cells/ml was selected two further parameters are displayed (Factor and Multiplier). The measured absorbance in these turbid samples is due to light scattering, and not the result of molecular absorption. A calibration curve can be determined by comparing measured OD 600 to expected OD 600 determined by using an alternative technique (e.g. microscope slide method).

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