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The following points highlight the two methods used for extraction of plant genomic DNA. The methods are: 1. CTAB Method 2. Potassium Acetate Method.
1. CTAB Method:
Plant genomic DNA is required mainly for mapping and isolation of genes for genetic engineering. If the protocol is good, pure and intact DNA can be obtained. The procedure should be adequate, quick and simple and also cost-effective. For extraction of DNA from organelles, material should be ground with osmaticum at 4°C.
Principle:
Cell wall and cell membranes of plant cells are removed one by one (lyse the cells gently) using an extraction buffer (CTAB). Bulk impurities like carbohydrates, contaminating proteins, RNAs and other molecules are removed by adding potassium acetate and DNA is precipitated with isopropyl alcohol.
Requirements:
Reagents:
1. Extraction Buffer (2x CTAB) 500 ml:
Tris HCl (pH 8.0) – 100.0 mM
NaCl – 1.4 mM
EDTA (pH 8.0) – 20.00 mM
CTAB – 2.0% (W/V)
2-Mercapto ethanol – 0.1% (W/V)
Extraction buffer should be autoclaved at 15 lb pressure for 15 minutes without mercapto-ethanol and this should be added only before use.
2. Liquid Nitrogen.
3. TE Buffer (10:1):
Tris HCl (pH 8.0) – 50.0 mM
EDTA – 10.0 mM
SDS – 20%
4. Chloroform: isoamyl alcohol (24:1).
5. Isopropanol.
6. Ethanol 70% (ice cold).
7. Sodium acetate (pH 5.2).
8. RNAse A solution (10 mg/ml).
9. Absolute alcohol.
10. Rice leaves.
11. Mortar and pestle.
12. Centrifuge and tubes.
13. Eppendorf tubes.
14. Pasteur pipettes and tops.
15. Vortex mixer.
16. Water-bath set at 60°C and 90°C.
17. Spatula.
Procedure:
1. Wash rice leaves in running tap water followed by sterile distilled water, blot with filter paper, cut the leaves into pieces and weigh 3—5 g.
2. Grind the leaf pieces to a fine powder with liquid nitrogen. Do not allow the powder to thaw.
3. Transfer the frozen powder by a spatula to a 50 ml tube (conical flask) containing 15 ml 2x CTAB extraction buffer with mercaptoethanol maintained at 60°C in a water bath and keep it there for 30 minutes with intermittent gentle shaking.
4. Cool the mixture to room temperature and add equal volume of chloroform: isoamyl alcohol (24:1) and mix gently by inverting the tube several times.
5. Centrifuge the content at 12000 rpm for 5 minutes and take out the aqueous layer carefully.
6. To this aqueous layer which contains DNA, add isopropanol (0.6 volume). Mix carefully by tilting the tube gently and keep at -20°C for 20-25 minutes to precipitate the DNA.
7. Either hook out DNA with a Pasteur pipette with curved tip to a new set of tubes or pellet DNA by centrifuging at 12000 rpm for 15 minutes.
8. Wash hooked DNA/pellet with ice cold 70% ethanol and air-dry the pellet.
9. Suspend DNA pellet in minimum amount of TE buffer (upto 2 ml depending on the size of pellet).
Purification:
1. Add 10 µl of RNAse (10 mg/ml) to DNA solution and incubate at 37°C for 30 minutes to remove RNA impurity.
2. Add equal amount of chloroform: isoamyl alcohol (24: 1), mix well and centrifuge at 12000 rpm for 5-8 minutes.
3. Add 1/10 volume of 3(M) sodium acetate and 2.5 volumes of absolute alcohol and mix gently. DNA precipitates.
4. Incubate at -20°C for 1 hour to increase precipitation and yield of DNA.
5. Hook DNA precipitate by a Pasteur pipette with curved tip or collect by centrifugation and wash with 70% ethanol.
6. Resuspend in T.E. buffer. Store at 4°C for few weeks or freeze at -20°C for longer use.
2. Potassium Acetate Method:
Principle:
Grinding of leaf tissue with liquid N2 helps to remove cell wall and the use of Sodium Dodecyl Sulphate (SDS), a detergent, in the extraction buffer disturbs cell membrane and releases cell contents. Potassium acetate selectively precipitates pigments, tannins etc. present in the cell.
Ethylene Diamine Tetra Acetic acid (EDTA) will chelate the Mg ion which is an essential co-factor for the enzyme to act. Thus it prevents the endonucleases to act on nucleic acid. Β mercaptoethanol is a reducing agent which protects DNA against quinones, disulphides, peroxidases and polyphenol oxidases.
Requirements:
1. Reagents
2. Extraction buffer:
NaCl – 500 mM
Tris HCl (pH 8.0) – 100 mM
EDTA (pH 8.0) – 50 mM
2-Mercaptoethanol – 10 mM
Add 2-3 µl of mercaptoethanol prior to use.
3. Potassium acetate stock 5 (M) and Sodium acetate 3(M).
4. High salt TE (IX):
Tris HCl (pH 8.0) – 50 mM
EDTA – 10 mM
SDS – 20%
5. Isopropanol (ice cold).
6. Liquid N2.
7. RNAse (sterile).
8. Mortar and pestle.
9. Water bath 65°C.
10. Centrifuge and tubes.
11. Rice leaves.
12. Absolute alcohol-ice cold.
Procedure:
1. Grind 2—3 g of rice leaves with liquid nitrogen after washing and drying the leaves.
2. Transfer the frozen powder to a 50 ml tube with 10 ml extraction buffer.
3. Add one ml of 20% SDS to the tube and incubate in a water bath at 65°C for 30 minutes by shaking it gently.
4. Add 5 ml of potassium acetate (5M), mix gently and incubate on ice for 20- 30 minutes.
5. Centrifuge at 5000 rpm for 15-20 minutes at 4°C. Filter supernatant that contains DNA.
6. Add 10 ml of ice cold isopropanol, tilt the tube and mix gently. Incubate at -20°C for half an hour or spool out or hook out DNA.
7. Add 500-600 of TE buffer and dissolve DNA.
Purification:
8. Add 7.0 µl RNAse and incubate at 37°C for 20-30 minutes.
9. Add 1/10 volume of 3(M) sodium acetate and twice the volume of ice cold absolute alcohol. DNA precipitates. Leave overnight at —20°C.
10. Centrifuge at 12,000 rpm for 10 minutes. Dry and dissolve the pellet in T.E. Store at -20°C.
11. Check by loading in 1% agarose gel.