jenway 6850 Operating Manual

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jenway 6850 Operating Manual Manual pdf 15 page image

153.2 NUCLEIC ACID DETERMINATIONDNA, RNA and oligonucleotides can be measured directly in aqueous solutions in a diluted orundiluted form. Aqueous buffers with low ion concentrations (e.g. TE buffer) are ideal for this method.The concentration is commonly determined by measuring at 260nm against a blank and thenevaluating against a factor.The 6850 has pre-defined methods installed which assume that absorption of 1 OD (A) is equivalentto, approximately: 50μg/ml dsDNA, 37μg/ml ssDNA, 40μg/ml RNA and 30μg/ml for oligonucleotides.DNA interference by contaminants can be assessed by the calculation of an absorption ratio. Theratios A260/A280 and A260/A230 are used to estimate the purity of nucleic acids, since proteinsabsorb at 280nm and substances such as peptides, phenols, aromatic compounds or carbohydratesabsorb at 230nm. Pure DNA should have an A260/A280 ratio of approximately 1.8 and pure RNA 2.0.In pure nucleic acid samples the A260/A230 ratio should be approximately 2.2.Nucleic acid concentration can also be estimated with the following calculations:Conc (μg/ml) = (Abs@260nm x 62.9) – (Abs@280nm x 36.0)Conc (μg/ml) = (Abs@260nm x 49.1) – (Abs@230nm x 3.48)Referring to a blank value where no absorption should occur is commonly required. On the 6850 thedefault reference wavelength is 320nm and the user can include the measured absorbance value inall nucleic acid calculations. The default wavelength can be modified from 320nm if required.3.3 SPECTROSCOPY MEASUREMENTThere are four main components of a spectrophotometer. These are a light source to emit a high andconstant amount of energy over the full wavelength range; a method for separating the light intodiscreet wavelengths; a sample holder and a light detector.The optical layout of the 6850 spectrophotometer is shown overleaf:The light from the tungsten and deuterium lamps is focused onto the grating which separates the lightinto discreet wavelengths. The diffracted spectrum of light then passes through a further slit and lensarrangement before passing through a beam splitter which directs half of the light towards to sampleholder and half towards the reference sample holder. The light which is not absorbed by the twosolutions is transmitted through a collecting lens and onto the signal detector. The signal from eachphoto-diode detector is used to calculate the % transmittance. The result is displayed either as %transmittance or absorbance on the instrument display.