Removal of melanin

Method is performed according to [Lagonigro et al].

Principle is that CTAB is charging the anionic nucleotides whereby neutral polysaccharides/ melanins are remaining in supernatant. This method also uses urea with the idea that the presence of urea helps to solubilize hydrophobic compounds that would otherwise potentially interact with the hydrophobic core of the CTAB micelles.

Solutions:

  • 5M NaCl
  • CTAB-Urea buffer
    • 50 mM Tris-HCl, pH 7.0
    • 1% CTAB
    • 4M Urea
    • 1 mM EDTA
  • 7M guanidine hydrochloride

Procedure:

  1. Water is added to ~ 100- 200 µL DNA/RNA solution until a volume of 400 µL is reached.
  2. 130µL 5M NaCl is added and 1.6 mL of CTAB-Urea solution
  3. Samples are mixed (by hand) and incubated o/n at 4°C
  4. Samples are centrifuged for 15 minutes at max speed at 4°C
  5. Precipitated RNA/DNA is resuspended in 400 µL 7M guanidine hydrochloride
    This is a critical step! Not all DNA will form a pellet at the bottom, but it will also adhere to the side of the tube and some will float on top of the solution. Be very careful in removing the solution! During this removal it is probably needed to perform additional centrifugation steps to reduce risk of losing DNA.
  6. Add 2 Vol of EtOH (100%)
  7. Incubate on ice for 1 hour
  8. Centrifuge for 15 min at 4 °C
  9. Wash twice with 70% EtOH
  10. Centrifuge 10 min at max speed at RT
  11. Remove supernatant, dry pellet an resuspend in TE
  1. Lagonigro MS, De Cecco L, Carninci P, Di Stasi D, Ranzani T, Rodolfo M, Gariboldi M: CTAB-Urea method purifies RNA from melanin for cDNA microarray analysis. Pigment Cell Res 2004, 17:312-315. DOI: 10.1111/j.1600-0749.2004.00155.x

Contributed by Pedro Crous, Janneke Bloem

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