Spectrophotometric Determination
Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents. The ratio of OD260/OD280 should be determined to assess the purity of the sample. This method is however limited by the quantity of DNA and the purity of the preparation. Accurate analysis of the DNA preparation may be impeded by the presence of impurities in the sample or if the amount of DNA is too little. In the estimation of total genomic DNA, for example, the presence of RNA, sheared DNA etc. could interfere with the accurate estimation of total high molecular weight genomic DNA.
Procedure
1. Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as 280nm.
2. Add 10 ml of each DNA sample to 900ml TE (Tris-EDTA buffer) and mix well.
3. Use TE buffer as a blank in the other cuvette of the spectrophotometer.
4. Note the OD260 and OD280 values on spectrophotometer.
5. Calculate the OD260/OD280 ratio.
Comments:
Ø A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.
Ø A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.
Ø A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol. In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA.
6. The amount of DNA can be quantified using the formula:
DNA concentration (mg/ml) = OD260 x 100 (dilution factor) x 50 mg/ml
1000
Spectrophotomteric Conversions for Nucleic Acids:
1 A 260 of ds DNA = 50 mg/ml
1 A 260 of ss oligonucleotides = 33 mg/ml
1 A 260 of ss RNA = 40 mg/ml
Reference
Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols: CIMMYT Applied Biotechnology Center Mexico
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