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DNA Quantification Protocol

DNA Quantification Protocol

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DNA Quantification and Quality Analysis
After isolation of DNA, quantification and analysis of quality are necessary to ascertain theapproximate quantity of DNA obtained and the suitability of DNA sample for furtheanalysis. This is important for many applications including digestion of DNA by restrictionenzymes or PCR amplification of target DNA. The most commonly used methodologies for quantifying the amount of nucleic acid in a preparation are: (i) gel electrophoresis; and (ii)spectrophotometric analysis. If the sample amount is less, the former method is usually preferred.
A. Agarose Gel Electrophoresis for DNA Quantification and Quality Analysis
This method of quantification is based on the ethidium bromide fluorescent staining of DNA.Ethidium bromide is a fluorescent dye, which intercalates between the stacked bases. Thefluorescent yield of the dye:DNA complex is much greater than the unbound dye. UVirradiation at 254nm is absorbed by the DNA and transmitted to the dye and the bound dyeitself absorbs radiation at 302nm and 366nm. This energy is retransmitted at 590nm, thereddish-orange region of the visible spectrum. In case of plant genomic DNA, the nucleicacids are electrophoretically separated on a 0.7-0.8% agarose gel containing ethidium bromide at a final concentration of 0.5
µ
g/ml. The quantity of DNA can be estimated bycomparing the fluorescent yield of the samples with a series of standards, for instance, lambda(
λ
) DNA at varying known concentrations. This provides a very rapid and sensitive means of estimating the nucleic acid concentration. A large number of samples with as little as 1-5ng of DNA can be quantified. Besides quantification, it also allows provides the advantage of analyzing the quality of the DNA preparation. Native DNA, which migrates as a tight band of high molecular weight (
40 kb), presence of RNA, and degraded/sheared DNA, if any, can be visually identified on the gel.
Procedure 
1.Prepare a 0.8% agarose gel.
2.
Add 1
µ
l of 6X gel loading dye to 2-3
µ
l of each DNA sample before loading the wellsof the gel. Addition of dye allows us to note the extent to which the samples might havemigrated during electrophoresis, so that it can be halted at an appropriate stage.
3.
Load at least 1 or 2 wells with uncut, good quality
λ
DNA or any previously quantifiedDNA samples (50ng and 100ng) as molecular weight standards.4.Run the submarine electrophoretic gel at 70V till the dye has migrated one-third of thedistance in the gel.5.DNA can be visualized using a UV transilluminator and quantified in comparison with thefluorescent yield of the standards.
Note:
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 analysisrequires larger quantities of DNA, since the technique is not PCR-based.
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. Purines and pyrmidines in nucleic acid show
 
absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if theDNA sample is pure without significant contamination from proteins or organic solvents. Theratio of OD
260
/OD
280
should be determined to assess the purity of the sample. This method ishowever limited by the quantity of DNA and the purity of the preparation. Accurate analysisof the DNA preparation may be impeded by the presence of impurities in the sample or if theamount 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 highmolecular weight genomic DNA.
Procedure
1.Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as280nm.
2.
Add 10
µ
l of each DNA sample to 900
µ
l TE (Tris-EDTA buffer) and mix well.3.Use TE buffer as a blank in the other cuvette of the spectrophotometer.
4.
 Note the OD
260
and OD
280
values on spectrophotometer.
5.
Calculate the OD
260
/OD
280
ratio.
Comments:
A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleicacids.
A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.
A ratio higher than 2.0 indicates that the samples may be contaminated withchloroform 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 (
µ
g/ml) = OD
260
x 100 (dilution factor) x 50
µ
g/ml1000
Spectrophotomteric Conversions for Nucleic Acids:
1 A 260 of ds DNA = 50
µ
g/ml1 A 260 of ss oligonucleotides = 33
µ
g/ml1 A 260 of ss RNA = 40
µ
g/ml
Reference
Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols:CIMMYT Applied Biotechnology Center. Second Edition, Mexico, D.F.: CIMMYT.
Laboratory for Microbial Ecology
 
Department of Earth, Ecological and Environmental SciencesUniversity of Toledo
DNA quantification (spectrophotometry)
Materials
DNA standard solutions
We have a standard series of herring sperm DNA solutions that includes DNA concentrationsof 500, 100, 50, and 10 ng DNA μl
-1
. Keep stocks of these solutions by diluting theconcentrated herring sperm DNA (10 mg ml
-1
) accordingly in DNase/RNase-free water.D.I. water for dilution of DNA prior to reading.One sterile 1.5 ml microcentrifuge tube for each samplePlastic (for concentrated samples, >50 μg ml
-1
) or quartz (for low concentration samples, < 50μg ml
-1
) cuvettes.Spectrophotometer • Wear gloves throughout the entire protocol.• Do not cross-contaminate your samples or the solutions. Be aware of your pipette tip.• Work clean, either on fresh blue bench paper, in the hood, or on a freshly ethanol treated bench top.• Perform all centrifugations with the hinge of the tube pointing “up”.• Do not use a vortex at any point in this protocol unless specified.
Begin by turning on the spec to allow the lamp to warm up for approximately 15 minutesbefore reading samples. Be sure that there is no cuvette in the well as the spec goesthrough its self-diagnosis.
 The protocol in brief 
You will quantify DNA in solution by measuring the absorbance of light (260 nm) in aspectrophotometer.
A. Preparing DNA samples
1. Remove the standards and your samples from the freezer and thaw them
on ice or in therefrigerator
. Mix them by tapping the side of the tube with your finger. Do not vortexto mix.2. In a separate sterile 1.5 ml microcentrifuge tube for each standard/sample, mix 10 μl of DNA with 990 μl of D.I. water. Vortex to mix. Let this solution stand for 10 minutesto ensure the complete diffusion of DNA throughout the solution. This represents a1:100 dilution of the standards and your DNA samples.
B. Preparing the spectrophotometer
A typical spectrophotometer 
3. Select “DNA/RNA”, and follow the cues given by the spec to set the machine up for your readings:We most commonly quantify: dsDNA

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Vishwajeet Chavan added this note
As you have given the formula, DNA concentration (µ g/ml) = OD 260 x 100 (dilution factor) x 50 µ g/m /1000. What is reason to divide the formula by 1000?
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