DNA isolation

DNA Extraction with CTAB buffer for amplified fragment length polymorphism (AFLP) and sequencing of PCR-amplified markers

Protocol

• Grind 100 mg tissue in a mortar in liquid nitrogen
•  pour leaf powder into a centrifuge tube 
• After liquid nitrogen has evaporated, add 3.5 ml CTAB extraction buffer, shake gently and collect tubes on ice 
• Add beta-Mercaptoethanol (4 µl/tube) 
• 30 min at 60°C in water bath 
• Optional: centrifugation at ca. 6000 g for 10 min, keep supernatant 
• Add 3 ml Chloroform:Isoamyl-alcohol (24:1) to supernatant, shake vigorously (but avoid vortexing!) 
• Centrifuge at ca. 6000 g for 10 min, at room temperature 
• Collect aqueous phase with cut off pipet tip 
• Add 2.7 ml isopropanol for precipitation 
• Centrifuge at ca. 6000 g for 10 min, at room temperature 
• Wash pellet thrice with 2 ml 70% ethanol (pellet should swim in alcohol) 
• Centrifuge at ca. 6000 g for 10 min and discard liquid 
• Dry pellet at room temperature over night 
• Dissolve in 50 µl water with non-acidic pH and transfer into Eppendorf tube.

In addition, for further analysis by AFLP it is suggested to digest RNA. For PCR amplification followed by fragment sequencing it is not required but improves the precision of DNA quantification by UV (260 nm) measurement.

Optional: RNA digestion

• Dilute RNase stock solution (100 mg/ml) 1:20 with water and add 1 µl to each sample 
• 30 min at 37°C in water bath 
• Add 49 µl water, 10 µl 3 M Natriumacetate, 300 µl isopropanole and mix thoroughly (no vortexing) 
• Incubate for 30 min at 4°C 
• Centrifuge ca. 20,000 g for 20 min at 4°C 
• Discard liquid 
• Add 800 µl 70% ethanol (icecold) 
• Centrifuge ca. 20,000 g for 20 min at 4°C 
• Discard liquid 
• Dry pellet at room temperature over night (optional: vacuum concentrator for 5-15 min) 
• Add 50 µl water (optional: 50 µl TE-buffer)

CTAB buffer (adjusted to pH 8.0)

TE buffer (pH 8.0-9.0)

Reference: Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19: 11-15