Plant Genomic DNA Extraction
Aim: To isolate genomic DNA from plant cells.
Principle: In order to recover DNA from plant cells, cell
wall should be digested by using detergent i.e. CTAB. EDTA present in
extraction buffer chelates metal ions needed for DNase and so inhibit the
activity of enzyme. Protein is extracted by treatment with distilled phenol.
Phenol also helps in the isolation and recovery of DNA from the aqueous phase.
A small amount of isoamyl alcohol serves as anti foaming agent. After removal
of the protein, DNA is purified by precipitation with ethanol. Incubation with
RNase ensures that contamination of DNA by RNA is kept at a minimum level. Tris
maintains the pH.
CTAB[Cetyl Tri Methyl Ammonium Bromide] is a
cationic detergent that has the property of precipitating nucleic acids and
polysaccharides from solution of low ionic strength. It also enhances the rate
of renaturation of complementary DNA strands and stabilizes double stranded DNA
helix. Salts like NaCl and Sodium Acetate partially mask the negative charge of
DNA and aggregate it together. Beta- Mercaptoethanol prevents the oxidation of
phenolic compounds in the tissue and Poly Vinyl Pyrrolidone prevents the
ionization of phenolic compounds. Isopropanol and Absolute alcohol remove the
water molecules from DNA so that the DNA precipitates out of solution. 70%
ethanol removes the salt contaminants from the DNA pellet.
Requirements:
1. Extraction buffer- 100 mM Tris-HCl
20 mM EDTA
2% CTAB
0.4 -2% mercaptoethanol
1% PVP [poly vinyl pyrrolidone]
2. RNase A (10 mg/ml)
3. 24:1 Chloroform: Isoamyl alcohol
4. Isopropanol {ice cold}
5. 5M NaCl
6. Absolute Alcohol{ice cold}
7. Phenol:Chloroform:Iso amyl alcohol[25:24:1]
8. 70% Ethanol(ice cold)
9. 3M Sodium Acetate
10. Tris-EDTA
Protocol :
1.
Crush 150 mg of fresh tomato leaves (healthy and infected) in 500 µl of
extraction buffer with the help of a pestle and mortar and then take in
eppendorf tubes.
2.
Incubate the samples at 50º C for 10 minutes. Then add 500µl of 24:1 Chloroform: Isoamyl alcohol. Keep the tubes in a shaker for 15
minutes.
3.
Centrifuge the samples at 10, 000 rpm for 12 minutes at room temperature.
Transfer the supernatant to fresh eppendorf tubes and add equal volume of
ice-cold isopropanol and 0.5 volumes of 5M NaCl, gently mix and precipitate DNA
by incubating the tubes at -20 º C for 2-3 hours.
4. Recover DNA by centrifuge at 10000 rpm for 12
minutes at 4º C.
Air
dries the pellet and resuspend in 500µl of 1x TE buffer.
5. Add 8 µl
of RNase A, mix well and incubate at 37º C for 30 minutes.
6.
Following the incubation, add equal volume of phenol: chloroform: isoamyl
alcohol and centrifuge at 10, 000 rpm for 12 minutes at room temperature. Transfer
supernatant to fresh eppendorf tubes.
7.
To the supernatant add equal volume of chloroform: isoamyl alcohol (24:1)
and centrifuge at 10, 000 rpm for 12 minutes at room temperature.
8. Then re-precipitate the DNA by adding 0.1 volume of 3M sodium acetate and
2 volumes of absolute alcohol and incubate at -20 º C overnight.
9.
Recover DNA by centrifuge at 10, 000 rpm for 12 min at 4 º C and wash the
pellet with 1ml of 70% ethanol(i.e., spin at 12000rpm for 4 min).
10. Air dry
the pellet and resuspend in 50 ml of 1x
TE buffer.
11. Use 15 ml of the samples for loading on agarose gel.
Result:
Visualize the distinct DNA bands of DNA on 0.8% Agarose gel electrophoresis.
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