1. Introduction1.1. DNAPurity The DNA Purity indicates the extent towhich the DNA is pure.
The DNA isusually present along with phenols , proteins and other contaminants. So whenisolated , how pure will the DNA be without the contaminants.1.2. Needfor DNA Purity Various experiments require Nucleic Acidswith certain purity for optimum performance.Spectrophotometric Quantification and UVFluorescence Tagging are the two important methods to determine the DNAPurity.1.
3.Approaches for the determination of DNA Purity Though fluoroscence measurement iseasier, absorbance measurement is usually preferred. This is because it issimple and can be carried out with common available lab equipments such as UVSpectrophotometer , UV transparent cuvettes and the isolated DNA. Absorbancereadings are performed at 260nm and 280 nm. 2.
Review ofthe Literature3. Aim andObjectives3.1. AimTo determinethe purity of DNA isolated from Leguminous seeds such as Cajanus cajan (The Pigeon pea ) , Pisum sativum (The Pea) and Viciafaba (The Field bean ).3.2.Objectives? Toisolate the DNA from The Field bean , The Pea and The Pigeon pea.? Toassess the Absorbance using the UV Spectrophotometer.
? Tocalculate the purity of the isolated DNA.4. Materials and Method4.1. Sample collectionThe Samples ( pigeon pea , pea and field pea ) were purchasedfrom the local market at Vyasarpadi. The pods were removed and washed withdistilled water. The seed coats were removed and weighed using a weighingmachine. 4.
2. Reagents? CTABBuffer – was prepared by dissolving 3ml of 10% Cetyltrimethyl ammonium bromide(CTAB) , 2.8 ml of 5M Sodium chloride , 0.
4 ml of 0.5M EDTA (pH 8.0) , 1 ml ofTris – HCl (pH 8.0) , 0.3g of Polyvinylpyrrolidone and 0.
02 ml of ?-Mercaptoethanol in 2.48 ml of distilledwater.? Chloroform? Isoamylalcohol? 7.5Mammonium acetate – was prepared by dissolving 5.78g of ammonium acetate in 10ml distilled water.? Ethanol? TEbuffer – was prepared by adding 0.
5 ml of 1M Tris-Cl (pH 8.0) and 0.1 ml of0.5M EDTA and making up the volume up to 10 ml using distilled water.4.3.Isolation of DNA ? 200mgof the sample tissues was weighed.
? 500?l of CTAB Buffer was added and grinded using a mortar and pestle.? Themixture was transferred to the centrifuge tubes and was labelled.? Forincubation, these centrifuge tubes were kept in a water bath for about 5minutes at 55°C.? Thecontents of the centrifuge tubes were mixed well and centrifuged for 5 minutesat 10,000 rpm.? Thesupernatant were transferred to another centrifuge tube.
? Thepellets were discarded.? Tothe supernatant, 25 ?l of a mixture of chloroform and isoamyl alcohol in theratio of 24:1 was added and mixed thoroughly.? Themixture was centrifuged at 10,000rpm for about 2 minutes.
? Aftercentrifugation, three layers were obtained.? Theupper aqueous layer was transferred to another tube to which 50 ?l of 7.5Mammonium acetate was added.? 500?l of ice cold absolute ethanol was added.
4.4Analysis? 150 ?l of the isolated DNA wasresuspended in 850 ?l TE Buffer.? Absorbance was taken at 260 nm and280 nm.? The values were tabulated.
? The ratio of the absorbance at 260 nmto the absorbance at 280nm gives the purity of the DNA.4.5. Observation Table : 4.5.1.
Absorbance of the Isolated DNA at 260 nm and 280 nm. Samples Absorbance at 260 nm Absorbance at 280 nm A ( Vicia faba ) B (Cajanus cajan ) C (Pisum sativum )