CTAB TECHNIQUE / Method / Schedule / Protocol FOR DNA ISOLATION / DNA EXTRACTION FROM PLANT LEAF / LEAVES SAMPLES
(see also DNA RNA double isolation procedure if both DNA and RNA are needed)
Reagents needed
CTAB buffer
2% CTAB 20gm CTAB
20mM EDTA 40ml EDTA stock (0.5M)
100mM Tris-Cl pH 8.0 100ml Tris-Cl stock (1M)
1.4M NaCl 280ml NaCl stock (5M)
76% Ethanol
10mM NH 4 Ac
DNA Extraction
1. Preheat 5ml CTAB (add 10µl mercaptoethanol to each 5ml CTAB) in a blue-topped 50ml centrifuge tube at 60-65 o C. Remove and discard midribs, and wrap laminae in aluminium foil and freeze in liquid nitrogen. 0.5 ?1.0 gm tissue/5ml CTAB
(Can store leaf material after liquid Nitrogen ?1-2 days at ?0 or ?0 for longer periods)
2. Gently crumble leaf tissue over cold pestle of liquid nitrogen. Grind frozen leaf with one spatula of fine sand add 0.5 spatula of PVPP powder after grinding.
3. Scrape powder into dry tube and add pre-heated buffer and mix gently. Avoid leaving dry material around rim of tube. Adjust CTAB volume to give a slurry-like consistency, mix occasionally.
4. Incubate for 60 min at 60 ℃
5. Add equal volume of chloroform/iso-amyl alcohol (24:1), Mix for about 3min, then transfer contents to narrow bore centrifuge tubes. Balance by adding extra chlor/iso. Spin 5,000rpm for 10min (ensure correct tubes used), brake off. (For extra pure DNA isolation - spin and retain supernatant before chloroform extraction).
