What quantity and concentration from SDS , beta-mercuptoethanol and PVP in CTAB buffer for DNA extraction from date palm?
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1. Transfer alcohol dipped samples from micro centrifuge tubes to the tissue paper. Let
them dry for some time.
2. Transfer the same samples to the new pre-marked micro centrifuge tubes.
3. In this tube Add 600 μl of 60 °C pre-warmed CTAB (Cetyl trimethyl ammonium
bromide) + 2 % PVP (Polyvinylpyrrolidon).
4. To this Add 3 μl β-marcapto-ethanol.
5. Crush the samples in micro centrifuge tubes with the help of sterile scissor and pestle.
6. Vortex for 5 minutes.
7. Incubate for 1 hour at 55 °C in a dry bath. While incubating, vortex at the interval of 20
minutes.
8. After incubation Add 10 μl 10 % SDS (Sodium dodecyl sulphate) solutions.
9. Repeat step number 6.
10. Incubate for 2 ½ hour at 55 °C in a dry bath. While incubating, vortex at the interval of
20 minutes.
11. Centrifuge at 14000 rpm at room temperature for 10 minutes.
12. After centrifugation Add 600 μl Chloroform: isoamyl alcohol (24:1).
13. Repeat step number 6.
14. Repeat step number 11.
15. Transfer the supernatant in a fresh micro centrifuge tubes.
16. Add 400 – 500 μl chilled isopropanol to the supernatant and mix the solution gently till
white flakes appears. Gently invert the micro centrifuge tubes to mix the solution.
17. Keep micro centrifuge tubes at -50 °C for 1 hour.
18. Centrifuge at 14000 rpm for 10 minutes at 4 °C.
19. Decant the supernatant and add 70 % chilled ethanol to wash the pellet.
20. Centrifuge at 10000 rpm for 10 minutes at 4 °C.
21. Repeat step number 19.
22. Repeat step number 20.
23. Repeat step number 19.
24. Repeat step number 20.
25. Decant the supernatant and dry the pellet at room temperature for 5 hours.
26. Add 50 μl TE (Tris EDTA buffer) and let DNA dissolve for 2 hours by incubating in a dry
bath at 55 °C.
27. Proceed for electrophoresis and Nano-drop spectrometer.
28. Store DNA at -20 °C.
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