Isolation of DNA from available plant materials such as green pea seeds, spinach, papaya etc. | Biology Practical Class 12 | Gaurav Kumar

Isolation of DNA from available plant materials such as green pea seeds, spinach, papaya etc.

Aim:-

            Isolation of DNA from available plant materials such as green pea seeds, spinach, papaya etc.

REQUIREMENTS:-

Distilled water, spinach leaves, Tris, SDS, EDTA, isopropanol, sodium acetate, Chloroform, isoamyl alcohol and phenol of the analytical grade.

PROCEDURE:-

1.    The homogenization involves rupturing of cell wall and membranes; this product dispersing in the         buffer solution.

2.    The cell extract is obtained by either rupturing them mechanically or by lysing them chemically by         enzymes.

3.    Take around 0.5 gm of spinach leaf tissue in a mortar and grind the tissue with a pestle,                            homogenize it with 2 ml of extraction buffer.

4.    The extraction buffer (pH 8.0) consists of 100 mM tris, 20 mM EDTA, 0.5MNaCl, 7M Urea, 0.1%         β  - Mercaptoethanol and 2% SDS.

5.    Long fibers of the tissue were retained back after crushing and the homogenate was transferred to a         2 ml. microfuge tube.

6.    An equal volume of phenol chloroform (isoamyl alcohol 25 : 24 : 1) is added to the tubes and                mixed well by gently shaking the tubes.

7.    Before start, the experiment tubes are centrifuged at the room temperature for about 15 minutes at           15000 rpm. 

8.    The upper aqueous phase is collected in a new tube.

9.     An equal volume of chloroforms' (isoamyl alcohol 24 : 1) is added and mixed.

10.    The upper aqueous phase, obtained after centrifugation at room at room temperature for 10                     minutes at 15000 rpm is transferred to a ne tube.

11.    The DNA is precipitated from the solution by adding 0.1 volume of 3M. Sodium acetate pH 7.0             and 0.7 volume of isopropanol.

12.    After 15 minutes of incubation at room temperature the tubes were centrifuged at 4'C for 15                 minutes at 15000 rpm.

13.    The DNA pellet was washed with 100% ethanol and airdried. 

14.    The DNA was dissolved in TE (Tris-cl 10 nM, pH 8.0, EDTA 1mM).

15.    To remove RNA 5ul. of DNAs RNA (10 mg/ml) is added to the DNA.

16.    Concentration of DNA  sample can be achieved by ethanol precipitation in the presence of Na              and at a temperature of - 20'C or less.

17.    DNA obtained can be further used for PCR, DNA fingerprinting, genome mapping and                         recombinant DNA etc.

PRECAUTIONS:-

1.    The amount of leaf sample should be between 0.5 gm to 0.6 gm. There is a positive correlation                between the leaf weight in the range of 0.5 gm to 1.0 gm and DNA isolated.

2.    The chemicals needed for isolation of DNA should be manufactured by standard pharmaceuticals             viz rock chemicals, sigma, etc.

3.    Wash the plant material thoroughly with distilled water to remove any dust particles, blot dried and         weighed.