Isolate DNA from available plant material such as spinach, green pea seeds, papaya, etc

Aim: Isolate DNA from available plant material such as spinach, green pea seeds, papaya, etc.

Material/Apparatus Required:

• 95% ethanol
• Thermometer that will measure 60°C
• 250 mL beaker
• 1000 mL beaker (2)
• Funnel
• Test tube
• Pipets
• Freezer
• Refrigerator
• Hot tap water bath 55-60°C
• Ice water bath 4°C
• Measuring spoons
• Light colored dishwashing liquid/shampoo
• Table salt (1 tsp)
• Distilled water (90 ml)
• Spoon for mixing
• Large onions
• Sharp knife
• Filter paper

Principle:

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are two types of nucleic acid found in living organism DNA act as the genetic material in most of the organisms. Nucleic acid has 3 major components:

a) Nitrogen base: Purines (Adenine and Guanine) and Pyrimidines: (Cytosine, Thymine and Uracil)

b) Pentose sugar: Ribose (RNA) and Deoxyribose (DNA)

c) Phosphate group: nitrogen bases are linked to pentose sugar through N-glycosidic linkage. Two nucleotides are linked through 3'-5' phosphodiester linkage to form dinucleotide. DNA is made up of two polynucleotide chain, where the backbone is constituted by sugar-phosphate, and the bases project inside. Both polynucleotide chain has anti-parallel polarity. The process of isolating/ extracting DNA from a cell is the first step for many laboratory procedures in biotechnology. We must be able to separate DNA from unwanted substances of cell gently enough so that DNA is not broken up.

Procedure:

1. Put 95% ethanol in the freezer to chill it.
2. Prepare two water bath – one at 55-60°C and another filled with ice water around 4°C. For the hot water bath, use large metal pot can be used along with a thermometer with an appropriate temperature range. For ice bath, a mixing bowl filled with ice and water works well.
3. In the 250mL beaker, make a solution consisting of 10mL liquid dishwashing detergent/shampoo and ¼ (1.5 gm) of table salt.
4. Add distilled water to the beaker to make a final volume of 100mL. dissolve the salt by stirring. This is solution A.
5. a) The liquid detergent causes the cells membrane to break down and dissolve protein and lipid of the cells by disrupting the bond that hold the cell membrane together. The detergent cause lipid and protein to precipitate out. The table salt (NaCl) enables nucleic acid to precipitate out of the ethanol solution because it shields the negative phosphate end of the DNA, causing the DNA strand to come close together.
6. Put the 1000 mL beaker with chopped Plant tissue and solution in hot water bath at 55-60°C for 10-12 minutes.
7. a) Do not keep the mixture in hot water bath for more than 15 minutes because the DNA will begin to break down.
8. b) The heat treatment softens the phospholipids in the cell membrane and denature the DNase enzyme which if present, would cut the DNA into small fragment so that it could not be extracted.
9. Cool the mixture in an ice water bath, at about 4°C, as shown in figure, for 5 minutes. During this time, smash the tissue with spoon or available apparatus. This step slows down the breakdown of DNA.
10. Filter the mixture through a filter paper placed in a funnel over a new 1000 ml beaker. While filtering the tissue, try to keep foam away from getting into the filtrate.
11. Dispense the onion solution into fresh test tube with the volume not more than 5 ml.
12. Take the cold 95% ethanol and add it to the test tube to create an ethanol layer on the top of about 1 cm.
13. DNA is not soluble in layer. When ethanol is added to the mixture, all the components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the ethanol layer.
14. Let the solution stand for 2-3 minutes without disturbing it. it is important not to shake the test tube. Watch the white DNA precipitate out into the ethanol layer. When good results are obtained, there will be enough DNA to spool on to a glass rod. DNA has the appearance of white mucus.

Observation:

The white DNA precipitate out into the ethanol layer, while the components of the mixture, except for DNA, stay in the solution.

Result:

The DNA has been isolated and it appears as transparent, slimy, and white mucus.

Precautions:

1. Fresh plant materials (onion) should be used for extracting DNA.
2. Beakers, test tube and cups must be thoroughly cleaned and dried.
3. Handle ethanol carefully because it is highly flammable.