Aug 22, 2020

Public workspaceRemoval of Melanin V.2

DOI
dx.doi.org/10.17504/protocols.io.bj6zkrf6
  • 1UC Riverside
  • stajichlab
Jason E Stajich
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DOI: dx.doi.org/10.17504/protocols.io.bj6zkrf6
External link: http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0749.2004.00155.x/abstract
Protocol Citation: Jason E Stajich 2020. Removal of Melanin. protocols.io https://dx.doi.org/10.17504/protocols.io.bj6zkrf6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This is transcribed from work published in this study http://dx.doi.org/10.1111/j.1600-0749.2004.00155.x and http://1000.fungalgenomes.org/home/protocols/removal-of-melanin/
Created: August 22, 2020
Last Modified: December 14, 2020
Protocol Integer ID: 40889
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Abstract
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.
Materials
STEP MATERIALS
ReagentNaClSigma AldrichCatalog #53014
ReagentWater
ReagentGuanidine hydrochlorideSigma AldrichCatalog #G3272-1KG
Reagent70% ethanolFisher Scientific
Reagent70% ethanolFisher Scientific
Protocol materials
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
ReagentWater
ReagentGuanidine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #G3272-1KG
Reagent70% ethanolFisher Scientific
Reagent70% ethanolFisher Scientific
ReagentWater
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
ReagentGuanidine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #G3272-1KG
Reagent70% ethanolFisher Scientific
Reagent70% ethanolFisher Scientific

Add
ReagentWater
to ~Amount100 µL - Amount200 µL DNA/RNA solution until a volume of Amount400 µL is reached.

Add Amount130 µL Concentration5 Molarity (M)
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014

Add Amount1.6 mL mL of CTAB-Urea solution
Protocol
CTAB-Urea buffer
NAME
CTAB-Urea buffer
CREATED BY
Jason Stajich

50 mM Tris-HCl, pH 7.0
1% CTAB
4M Urea
1 mM EDTA
Mix samples (by hand).
Incubate overnight at Temperature4 °C
DurationOvernight
Centrifuge for Duration00:15:00 at max speed atTemperature4 °C
Remove the solution. Be very careful in this step!
Resuspended in Amount400 µL µL Concentration7 Molarity (M)
ReagentGuanidine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #G3272-1KG

Add 2Amount2 µL VOLUMES Vol of EtOH (100%)

Incubate on ice for
Duration01:00:00
Centrifigation15300 x g, 4°C, 00:15:00

Wash with
Reagent70% ethanolFisher Scientific

Wash again with
Reagent70% ethanolFisher Scientific

Centrifigation20800 x g, Room temperature, 00:10:00 , (or maximum speed)

Remove the supernatant.
Dry pellet
Resuspend in TE buffer