1. Introduction

1.1. DNA
Purity

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         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.

1.2. Need
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.

1.3.
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. 

2.
Review of the Literature

3. Aim
and Objectives

3.1. Aim

To
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 ).

3.2.
Objectives

?   
To
isolate the DNA from The Field bean , The Pea and The Pigeon pea.

?   
To
assess the Absorbance using the UV Spectrophotometer.

?   
To
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
machine.

4.2. Reagents

?     
CTAB
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
water.

?     
Chloroform

?     
Isoamyl
alcohol

?     
7.5M
ammonium acetate – was prepared by dissolving 5.78g of ammonium acetate in 10
ml distilled water.

?     
Ethanol

?     
TE
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.

 

4.3.
Isolation of DNA

?        
200mg
of the sample tissues was weighed.

?        
500
?l of CTAB Buffer was added and grinded using a mortar and pestle.

?        
The
mixture was transferred to the centrifuge tubes and was labelled.

?        
For
incubation, these centrifuge tubes were kept in a water bath for about 5
minutes at 55°C.

?        
The
contents of the centrifuge tubes were mixed well and centrifuged for 5 minutes
at 10,000 rpm.

?        
The
supernatant were transferred to another centrifuge tube.

?        
The
pellets were discarded.

?        
To
the supernatant, 25 ?l of a mixture of chloroform and isoamyl alcohol in the
ratio of 24:1 was added and mixed thoroughly.

?        
The
mixture was centrifuged at 10,000rpm for about 2 minutes.

?        
After
centrifugation, three layers were obtained.

?        
The
upper aqueous layer was transferred to another tube to which 50 ?l of 7.5M
ammonium acetate was added.

?        
500
?l of ice cold absolute ethanol was added.

4.4
Analysis

?      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.

 

4.5.
Calculation

A260/A280 ratio can be used as an estimate of DNA purity.

5. Result

Table : 4.5.1. Absorbance of
the Isolated DNA at 260 nm and 280 nm.

 

 

Samples

Absorbance
at 260 nm

Absorbance
at 280 nm

260:280
Ratio

A  ( Vicia faba )

2.908

1.304

2.23

B  (Cajanus cajan )

2.821

1.152

2.45

C  (Pisum sativum )

2.465

1.601

1.54

 

6. Discussion

For
DNA, Absorbance 260/280,

Between 1.6 – 2.0

purity level accepted

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.