May 20, 2020

Public workspaceSorbitol washing complex homogenate for improved DNA extractions

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.beuvjew6
  • 1Research School of Biology, Australian National University, Canberra, ACT, Australia
  • High molecular weight DNA extraction from all kingdoms
  • PLOS ONE Lab Protocols
Ashley Jones
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.beuvjew6
External link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0253830
Protocol Citation: Ashley Jones, Benjamin Schwessinger 2020. Sorbitol washing complex homogenate for improved DNA extractions. protocols.io https://dx.doi.org/10.17504/protocols.io.beuvjew6
Manuscript citation:
Jones A, Torkel C, Stanley D, Nasim J, Borevitz J, Schwessinger B (2021) High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. PLoS ONE 16(7): e0253830. doi.org/10.1371/journal.pone.0253830
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
We use this protocol and it's working
Created: April 09, 2020
Last Modified: May 20, 2020
Protocol Integer ID: 35445
Abstract
The extraction of pure DNA can be challenging due to the presence of sugars, oils and other endogenous chemicals present within biological samples. This is particularly true for many plants and fungi due to the presence of secondary metabolites such as polyphenols and polysaccharides. Polyphenols within the cytosol can become irreversibly DNA-bound after cell lysis and polysaccharides can co-precipitate with DNA during the extraction. Sorbitol is an osmotically active sugar alcohol and washing homogenate with sorbitol before cell lysis has been shown to significantly improve the purity of DNA extractions. Sorbitol does not pass cell membranes and likely acts by drawing the cytosol out of the cell. Therefore polyphenols and polyscacharides would be removed.
Guidelines
This protocol is based on the following publication. When citing, please also note the original publication below.

Inglis, P., Pappas, M.d.C., Resende, L. and Grattapaglia, D. (2018). Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications. PLOS ONE 13, 1-14.
Materials
Dithiothreitol (DTT) or β-Mercaptoethanol
EDTA pH 8
Milli-Q water (MQW)
PVP 40,000
D-Sorbitol
Tris-HCl pH 8
PREPARATION
PREPARATION
Prepare sorbitol wash solution. Approximately 2x volume of tube capacity will be used per sample. Sample tissue should not exceed 33% of tube capacity.

To prepare a 500 mL solution:

ReagentTarget concentrationMolecular weightStock concentrationAdd from stock
D-Sorbitol0.35 M182.17powder31.88 g
PVP 40,0001% (w/v)40,000powder5 g
Tris-HCl pH 8100 mM157.601 M50 mL
EDTA pH 85 mM292.240.5 M5 mL
MQWNANANABring to 500 mL

Note
  • Store at 4°C for up to 6 months.

  • DTT or β-Mercaptoethanol will be added freshly when used (below).

HOMOGENISATION
HOMOGENISATION
Grind tissue to a fine powder, keep frozen with liquid nitrogen.
If homogenate is not in a tube (i.e. mortar and pestle), transfer to an appropriate sized tube.
Note
Homogenate should not exceed 33% of tube capacity.

SORBITOL WASH
SORBITOL WASH
Fill the tube capacity with an excess of sorbitol wash solution.
Add DTT, approximate final concentration of 1 mM. Add 0.15 mg DTT per 1 mL of sorbitol wash. A tiny scoop of powder is sufficient. DTT MW = 154.253.
Note
Alternatively add 1% β-Mercaptoethanol (v/v). Add 10 µL of β-Mercaptoethanol per 1 mL of sorbitol wash.

Shake, invert and vortex to mix thoroughly.
Note
Ensure tissue is in suspension.

Centrifuge at 5,000 rcf for 5 min at room temperature.
Note
Limited based on tubes rupturing if they contain ball bearings. Can also be reduced to 2,500 rcf.

Carefully decant the supernatant. Remove as much of the wash solution as possible without losing the pellet.
Note
Supernatant should appear slightly cloudy or discoloured light yellow or brown.

If the supernatant was turbid, viscous or had dark discolouration, repeat the sorbitol wash.
DNA EXTRACTION
DNA EXTRACTION
Proceed to a DNA extraction method of choice by adding lysis buffer to the pellet. Continue protocol as normal.