The document describes a CTAB protocol for extracting genomic DNA from Gentiana tissue. The protocol involves grinding tissue and lysing cells using CTAB buffer at 65°C. RNA is removed using RNaseA. Chloroform-isoamyl alcohol is used to extract the aqueous phase containing DNA. Sodium acetate and ethanol are used to precipitate the DNA, which is then washed and resuspended in TE buffer.

1. CTAB Protocol for Gentiana Genomic DNA extraction
(by Ching-Sung G. Chang, JANUARY, 2018)
1. Grind frozen or dried tissue, add 1 mL of cold CTAB-free buffer + β-mercaptoethanol (β-ME can
be added as much to 2%).
2. Mix by inversion, keep 5-10 minutes on ice.
3. Spin at 10,000 g for 10 minutes, discard the supernatant. (If use particularly nasty tissue, repeat
step 1-3.)
4. Add 800 μL of pre-warmed 2× CTAB + 5-10 μL of β-ME, incubate at 65℃ for 60 minutes.
5. Let the tubes cool down and add 4 μL of RNaseA, stand at room temperature for 15 minutes.
6. Add 800 μL of chloroform-isoamylalcohol 24:1, shake completely for 3 minutes, then spin at
10,000 g for 10 min.
7. Transfer 700 μL of the aqueous phase to a new tube, add 700 μL of chloroform-isoamylalcohol
24:1, mix by inversions for 1 minutes.
8. Transfer 600 μL of the aqueous phase to a new tube, add 60 μL (
1
10
volume) of 3 M sodium
acetate, mix well.
9. Add 1320 μL (2× volume of aqueous phase) of ethanol, gently mix well. Leave on ice (or at
-20℃) for 30-60 minutes.
10. Spin at 10,000 g for 10 minutes, pour off the supernatant.
11. Wash pellet with 800 μL of 70% ethanol, Spin at 10,000 g for 3 minutes, pour off the supernatant.
12. Repeat wash with 99.5% ethanol.
13. Dry pellet and resuspend in 50-100 μL of 1× TE buffer. (or more, as preferred)
2× CTAB Buffer: (Per 200 mL)
100 mM Tris-HCl (pH 8.0)
1.4 M NaCl
20 mM EDTA
2% CTAB
2% PVP40
20 mL 1M Tris-HCl pH8.0
56 mL 5M NaCl (or 16.4 g)
16 mL 0.5 M EDTA
4.0 g
4.0 g