Genomic DNA Isolation From Plants By Modified CTAB Method
1. Plant leaves were freeze dried by liquid N2 and then grinded by morter-pestle (autoclaved and prechilled).
2. 0.2% of β-mercaptoethanol was added to prewarmed (65oC ) CTAB DNA isolation buffer.
3. 40ml of 1X CTAB solution was mixed with ground tissue.
4. Then the mortar-pestle was incubated at 65oC for 60 minutes with occasional stirring after every 10 mins.
5. After incubation, the mixture was distributed into eppendorfs and allowed to cool down.
6. To each eppendorf, an equal volume (750µl) of phenol: chloroform: isoamyl alcohol (25:24:1) solution was added and mixed by inversion (about 10 times).
7. Centrifuged at 12000 rpm for 15 minutes at room temperature.
8. The upper aqueous phase was transferred in a fresh eppendorf and the step was repeated once again.
9. 0.6 volume of isopropanol (180µl) was added to aqueous phase(300µl) in each eppendorf and mixed by inversion (about 10 times).
10. Incubated at -80oC for overnight.
11. Then the sample was centrifuged at 12,000 rpm for 20 minutes at 4oC.
12. The supernatant was removed.
13. DNA pellet was washed with 85% ethanol and centrifuged at 12,000 rpm for 10 mins at 4oC.
14. The supernatant was removed and the pellet was air dried.
15. Pellet was dissolved in sterile water.
16. After that RNase A (20µg/ml) was added and incubated for 30 mins at room temperature.
17. 0.3 volume CH3COONa and 2.5 volume of 98% ethanol was added and mixed by inversion.
18. Incubated at -80oC for overnight.
19. The sample was then centrifuged at 4oC for 20 mins at 12000 rpm.
20. Supernatant was discarded.
21. 70% ethanol (freshly prepared) was added and spun down for 15 mins at 4oC at 12,000 rpm.
22. Supernatant was removed and the pellet was dried.
23. Pellet was then dissolved in 2.5mM tris-HCl buffer.
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