Lichen DNA Extraction

DNA Extraction
This protocol describes a CTAB-based DNA extraction method, often used in plant DNA extraction, optimized for lichen tissue, which is rich in polysaccharides, polyphenols, and secondary metabolites that commonly inhibit DNA purification and downstream enzymatic reactions. By combining chemical lysis, selective precipitation, and organic extraction, the procedure isolates high-quality genomic DNA suitable for PCR amplification, sequencing, and molecular analysis. Special emphasis is placed on the removal of inhibitory compounds and contaminants to ensure reliable performance in downstream applications.

Procedure

CTAB disrupts membranes and complexes with nucleic acids, while EDTA chelates divalent cations to inhibit nucleases. PVPP binds polyphenols, which are abundant in lichen tissue and can inhibit downstream enzymatic reactions.

CELL LYSIS

Purpose: Break open cells and release DNA while neutralizing inhibitory compounds.

PVPP binds polyphenols released during cell disruption, preventing them from oxidizing and damaging DNA. Heat and agitation promote efficient lysis of fungal and algal cells.

Step 1. Add 1% w/v PVPP to lysis buffer. PVPP removes polyphenolic inhibitors.

Lichens contain high levels of secondary metabolites. PVPP is critical at this stage to prevent oxidative damage to DNA.

Step 2. Add 500 µL of lysis buffer to sample and shake vigorously.
Step 3. Incubate for 20 minutes at 70 °C, periodically mixing by shaking tube.
20 min @ 70 °C

REMOVE NON-POLAR MOLECULES

Purpose: Remove lipids, proteins, and other hydrophobic contaminants.

Chloroform denatures proteins and partitions non-polar molecules into the organic phase, leaving nucleic acids in the aqueous phase.

Chloroform is toxic, volatile, and a suspected carcinogen.

  • Always work in a certified fµme hood
  • Wear nitrile gloves and eye protection
  • Avoid skin contact and inhalation
Step 4. IN A FUME HOOD, add 500 µL of chloroform:isoamyl alcohol.
Step 5. Vortex briefly.
Step 6. Centrifuge for 3 min at 16,000 g.
3 min @ 16000 𝘨
Step 7. Carefully capture upper phase with a pipette and transfer into a new tube.

When pipetting the aqueous phase, leave a small volµme behind rather than risk disturbing the interphase.

CTAB PRECIPITATION

Purpose: Separate DNA from polysaccharides.

Under low-salt conditions, CTAB forms insoluble complexes with DNA while polysaccharides remain soluble, improving DNA purity.

Step 8. Add 1 ml of precipitation buffer, mix gently by inversion for 1 minute.
1 min
Step 9. Centrifuge for 10 minutes at 16,000 g
10 min @ 16000 𝘨
Step 10. Discard supernatant.
Step 11. Re-suspend pellet in 350 µL of 1.2 M NaCl.

REMOVE NON-POLAR MOLECULES

Purpose: Further purify DNA after CTAB precipitation.

A second chloroform extraction removes residual CTAB, proteins, and other contaminants released during resuspension.

Step 12. IN A FUME HOOD, add 500 μl of chloroform:isoamyl alcohol.
Step 13. Vortex briefly.
Step 14. Centrifuge for 3 min at 16,000 g
3 min @ 16000 𝘨
Step 15. Carefully transfer upper phase into a new tube.

ISOPROPANOL PRECIPITATION

Purpose: Concentrate and recover DNA.

Isopropanol reduces DNA solubility, allowing it to precipitate efficiently at lower volµmes and temperatures.

Step 16. Add 210 μl (0.6 volµmes) of cold isopropanol and mix carefully.
Step 17. Incubate for 10 minutes at -20 °C.
10 min @ -20 °C
Step 18. Centrifuge for 7 minutes at 16,000 g.
7 min @ 16000 𝘨
Step 19. Discard supernatant

FINAL ETHANOL WASH

Purpose: Remove residual salts and solvents.

An ethanol wash cleans the DNA pellet without dissolving it, improving downstream performance.

Step 20. Add 200 μl of 80% EtOH
Step 21. Centrifuge for 2 minutes at 16,000 g
2 min @ 16000 𝘨
Step 22. Discard supernatant.
Step 23. Dry pellet by inverting tube, cap open, on a paper towel.
Step 24. Be sure there is no residual EtOH before re-suspending in 50-100 µL sterile H₂O.