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The concentration of DNA that has been prepared can be determined with a spectrophotometer or by comparison with DNA standards on an agarose gel. DNA absorbs in the UV range of 260 nm. An optical density (O.D.) of 1.0 at 260 nm would indicate a concentration of 50 μg/ml of double stranded DNA.
The purity of DNA is determined by the ratio or OD260/OD280 = 1.8 - 2.0. the closer this figure, the purer the DNA sample and the more amenable it is to digestion. <1.8 would indicate contamination.
Part 1B - Agarose gel electrophoresis
Electrophoresis through agarose or polyacrylamide gels is the standard method that is used to separate, identify and purify DNA fragments. The technique is simple to perform and capable of resolving fragments of DNA that cannot be separated adequately by other procedures such as centrifugation.
Agarose gel is cast by melting the agarose in the presence of the desired buffer until a clear transparent solution is achieved. This solution is then poured onto the mould onto which combs have been placed and allowed to set/harden. The gel is positioned in a tank and covered in the casting buffer of choice.
The DNA fragments/species are able to migrate through a gel matrix across an electric current with a particular voltage that is applied, thus enabling the separation of different sizes according to distance of migration. DNA that is negatively charged at neutral pH migrates toward to anode (+). The larger and more spread out the piece of DNA, the slower the migration.
The rates of migration depend on several factors which include:
Direction of electric field
Setting up the restriction digests
A series of restriction enzymes can be used to digest the DNA. Digests are usually done in as small a volume as possible depending on the amount of DNA to be cut. 2 μg is usually sufficient for a digestion volume of 20 μl.
Theoretically 1U of restriction enzyme is sufficient to digest 1 μg of DNA at 37OC (or at the optimal temperature for the particular RE) for 1 hour. In practice up to a 10 fold increase of the enzyme is used to ensure complete digestion of the DNA within the time frame allocated.
Particular Res will be used for digesting the plasmid. The digestion is done under optimal conditions using the appropriate buffer for the particular enzyme used.
As stated in the practical manual.
As stated in the practical manual.
260nm = 0.4279
280nm = 0.2119
Ratio (OD260/OD280) = 2.0193
Conc. of DNA solution:
(Diluted DNA solution)
1.0 OD ~ 50 μg/ml
0.4279 OD ~ 50 x 0.4279 = 21.395 μg/ml
(Original DNA solution)
21.395 x 250 = 5348.75 μg/ml
The results of the band appear to be a smear.
[For the results of agarose gel electrophoresis, please refer to the attached paper]
The contents are stored for further use in practical 4.
Quantifying DNA is a technique to calculate the quantity (weight) of DNA in a sample but not for determination of the length (size) of a DNA band.
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 digestion of DNA by restriction enzymes or PCR amplification of target DNA. The most commonly used methodologies for quantifying the amount of nucleic acid in a preparation are gel electrophoresis and spectrophotometric analysis. If the sample amount is less, the former method is usually preferred.
Purines and pyrmidines in nucleic acid show maximum absorption around 260nm (dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents.
The efficiency of enzymatic reactions is dependent on the concentration of all components which includes the DNA template in PCR. Template DNA may be the source of potentially deleterious contaminants. Too much DNA binds up available Mg++ and too little DNA leads to insufficient final product.
When calculating the OD260/OD280 ratio, the range of value indicates as follows:
A ratio 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.
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.
The result of the electrophoresis appears to be a smear as it seems many bands are so close together that distinguishing between adjacent bands is not quite possible. This may be cause by contamination of stray nuclease that degraded the DNA into vast small pieces or it was over pipetted/vortexed too much during extraction which may cause DNA to shear into smaller bits.
The method for DNA quantification and quality analysis is learnt.