DNA Quantification and Quality Analysis by Spectrophotometry
Objectives:
1. To determine quantity of DNA in extract solution.
2. To determine purity of extracted DNA sample with respect to protein and RNA contaminants.
Theory:
After isolation of DNA, quantification and analysis of quality are necessary to ascertain the approximate quantity of DNA obtained and the suitability of DNA sample for further analysis. This is important for many applications including gel electrophoresis, digestion of DNA by restriction enzymes or PCR amplification of target DNA (For SSR and RAPD analysis, it is more important to have good quality DNA samples (unsheared/undegraded DNA), than high quantities of DNA. In contrast, RFLP analysis requires larger quantities of DNA, since the technique is not PCR-based). One of the most commonly used methodologies for quantifying the amount of nucleic acid in a preparation is spectrophotometric analysis.
B. Spectrophotometric Determination
Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Nitrogen bases in nucleic acid; Purines and pyrmidines show absorption maxima around 260nm (eg. dATP: 259nm; dCTP: 272nm; dTTP: 247nm; dGTP: 253nm and dUTP: 263) 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.
Spectrophotomteric Conversions for Nucleic Acids:
1 A 260 of ds DNA = 50 mg/ml
1 A 260 of ss oligonucleotides = 33 mg/ml
Requirements:
- Spectrophotometer
- Quartz cuvette
- DNA Sample
- TE Buffer (pH-8.00, Tris-10mM and EDTA 1mM)
- Micropipettes
Procedure
1. Take 3 ml TE buffer in a quart cuvette and calibrate the spectrophotometer at 260nm as well as 280nm.
2. Use TE buffer as a blank in the other cuvette of the spectrophotometer.
3. Add 50 ml of each DNA sample to 2950ml TE (Tris-EDTA buffer) and mix well.
4. Note the OD260 and OD280 values on spectrophotometer.
5. Calculate the quantity of DNA by using formula below.
6. Calculate the OD260/OD280 ratio.
7. The amount of DNA can be quantified using the formula:
DNA concentration (mg/ml) = OD260 x 60 (dilution factor in above) x 50 mg/ml
1000
Observation:
Table1: Absorbance of DNA samples at 260nm and 280nm
S. no. | DNA samples | OD260 | OD280 | DNA concentration | OD260/OD280 | Remarks |
1. | ||||||
2. | ||||||
3. | ||||||
4. |
Comments:
- A ratio in between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.
- A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers (Aromatic amio acids Tyrosine and Tryptophan have absorbtion maxima at 280 nm).
- A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol.
Note: In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA. For the removal of protein, repeat the protein removal step; phenol-chloroform treatment and in case of phenol and chloroform contaminants, re-precipitate the DNA with ethanol or isopropanol and dissolve in TE buffer. Repeat if further needed.
Reference
1. Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols: CIMMYT Applied
2. Tiwari K. B. and Ghimire P. (2010) A Practical Handbook for Microbial Genetics and Molecular Biology, First Edition, Kantipur College of Medical Sciences, Kathmandu.
0 comments:
Post a Comment