The DNA Purity indicates the extent to
which the DNA is pure. The DNA is
usually present along with phenols , proteins and other contaminants. Thus when
isolated , how pure will the DNA be without the contaminants.
for DNA Purity
Various experiments require Nucleic
Acids with certain purity for optimum performance. Spectrophotometric Quantification and UV Fluorescence Tagging are the two important methods to determine
the DNA Purity.
Approaches for the determination of DNA Purity
Though fluoroscence measurement is
easier, absorbance measurement is usually preferred. This is because it is
simple and can be carried out with common available lab equipments such as UV
Spectrophotometer , UV transparent cuvettes and the isolated DNA. Absorbance
readings are performed at 260nm and 280 nm.
Review of the Literature
determine the purity of DNA isolated from Leguminous seeds such as Cajanus cajan (The Pigeon pea ) , Pisum sativum (The Pea) and Vicia faba (The Field bean ).
isolate the DNA from The Field bean , The Pea and The Pigeon pea.
assess the Absorbance using the UV Spectrophotometer.
calculate the purity of the isolated DNA.
4. Materials and Method
4.1. Sample collection
The Samples (field pea , pigeon pea and pea) were purchased
from the local market at Perambur. The pods were removed. The seeds were washed
with distilled water. The seed coats were removed and weighed using a weighing
Buffer – was prepared by dissolving 3ml of 10% Cetyltrimethyl ammonium bromide
(CTAB) , 2.8 ml of 5M NaCl , 0.4 ml of 0.5M EDTA (pH 8.0) , 1 ml of Tris – HCl
(pH 8.0) , 0.3g of Polyvinylpyrrolidone and 0.02 ml of ?-Mercaptoethanol in 2.48 ml of distilled
ammonium acetate – was prepared by dissolving 5.78g of ammonium acetate in 10
ml distilled water.
buffer – was prepared by adding 0.5 ml of 1M Tris-Cl (pH 8.0) and 0.1 ml of
0.5M EDTA and making up the volume up to 10 ml using distilled water.
Isolation of DNA
of the sample tissues was weighed.
?l of CTAB Buffer was added and grinded using a mortar and pestle.
mixture was transferred to the centrifuge tubes and was labelled.
incubation, these centrifuge tubes were kept in a water bath for about 5
minutes at 55°C.
contents of the centrifuge tubes were mixed well and centrifuged for 5 minutes
at 10,000 rpm.
supernatant were transferred to another centrifuge tube.
pellets were discarded.
the supernatant, 25 ?l of a mixture of chloroform and isoamyl alcohol in the
ratio of 24:1 was added and mixed thoroughly.
mixture was centrifuged at 10,000rpm for about 2 minutes.
centrifugation, three layers were obtained.
upper aqueous layer was transferred to another tube to which 50 ?l of 7.5M
ammonium acetate was added.
?l of ice cold absolute ethanol was added.
? 150 ?l of the isolated DNA was
resuspended in 850 ?l TE Buffer.
? Absorbance was taken at 260 nm and
280 nm with TE Buffer as the blank.
? The values were tabulated.
A260/A280 ratio can be used as an estimate of DNA purity.
Table : 4.5.1. Absorbance of
the Isolated DNA at 260 nm and 280 nm.
at 260 nm
at 280 nm
A ( Vicia faba )
B (Cajanus cajan )
C (Pisum sativum )
DNA, Absorbance 260/280,
Between 1.6 – 2.0
purity level accepted
< 1.6 contamination with Protiens > 2.0
contamination with RNA or Phenol
From the results, the DNA isolated from the Sample A and Sample C are
probably contaminated with RNA or Phenol. The DNA isolated from Sample B are
probably contaminated with Protiens.
The abnormal ratios are because the DNA is not
the only molecule that can absorb UV light at 260nm. RNA and the aromatic amino
acids present in protein also has a great absorbance at 260nm. These factors affects
the value of absorbance. In addition , Carbohydrates are common contaminant related
to nucleic acid isolated from plant sources.