Non-destructive DNA extraction from DESS preservation solution

Eri Ogiso-Tanaka; Minako Abe Ito; Daisuke Shimada

Oct 24, 2024

Non-destructive DNA extraction from DESS preservation solution

This protocol is a draft, published without a DOI.

Eri Ogiso-Tanaka¹,
Minako Abe Ito²,
Daisuke Shimada¹

¹Center for Molecular Biodiversity Research, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, 305-0005, Japan;
²Center for Collections, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, 305-0005, Japan

Eri Ogiso-Tanaka

National Museum of Nature and Science, Japan

Protocol Citation: Eri Ogiso-Tanaka, Minako Abe Ito, Daisuke Shimada 2024. Non-destructive DNA extraction from DESS preservation solution . protocols.io https://protocols.io/view/non-destructive-dna-extraction-from-dess-preservat-cfeftjbn

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: August 17, 2022

Last Modified: October 24, 2024

Protocol Integer ID: 68775

Keywords: Specimens, Non-destructive DNA extraction, DNA barcoding, nematode, animal, diatom, COI, 18S, NGS, Nanopore, DESS, Sangar sequence, meiobenthos, preserve DNA and morphology

Funders Acknowledgements:

JSPS KAKENHI

Grant ID: JP21K06299

Abstract

DESS is a widely used storage solution to preserve DNA in biological tissue samples. DESS consists of 20% dimethyl sulfoxide (DMSO), 250 mM ethylenediaminetetraacetic acid (EDTA), and saturated sodium chloride (NaCl), and its efficacy has been confirmed in a variety of taxa and tissues. We introduce non-destructive DNA extraction and DNA barcoding using a portion of the DESS supernatant of a nematode specimen stored at room temperature for 10 years in DESS. This technique can also be used for the preservation and non-destructive DNA extraction of specimens of various species collected in the field. By immersing samples in DESS in the field to prevent DNA degradation, and then immersing them in new DESS in the laboratory after separation and identification to extract DNA from the supernatant, non-destructive DNA barcoding can be performed. Here, we provide full protocols on how to extract DNA from DESS solutions, and how to use the extracted DNA for DNA barcoding.

Materials

1.    DESS
1.1 Requirements for making DESS solution (ref: https://www.youtube.com/watch?v=ye_1FRIR8bY)
        Reagents
            ♦ Saturated sodium chloride (NaCl)
            ♦ 250mM EDTA (pH 8.0)            
            ♦ 20% DMSO                                  
            ♦ Deionized / Milli-Q water   
        Equipment and disposables
            ♦ Measuring cylinder/volumetric flask
            ♦ Conical flask / Beaker
            ♦ Magnetic stirrer
1.2 DESS preparation and recipe
        Equipment and disposables
                To prepare 1000 mL of DESS
            ♦ 500 mM EDTA (pH 8.0, 500 mL)
            ♦ DMSO (200 mL)
            ♦ NaCl (288 g)
Fill up to 1000 mL with sterile water.
Sterilize solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on a liquid cycle.
Store the DESS solution at room temperature (10~25˚C).
*Although NaCl may precipitate over time, this may not pose a problem; use the clear supernatant portion.

2.    DNA extraction
1.2 Requirements for DNA extraction 
        Reagents
            ♦ Silica (FUJIFILM: wakosil [232-00841])
            ♦ 0.01 mol/L Hydrochloric acid
            ♦ AMpure (Beckman Coulter) or SeraPure (refer to the method described below for the preparation )
            ♦ Ethanol (99.5%)
            ♦ Ethanol (75%)
            ♦ 500 mM EDTA (pH 8.0)
            ♦ 1 M Tris-HCl (pH 8.0) 
            ♦ Distilled water 
            ♦ TE buffer (pH 8.0) 
            ♦ 0.1× TE buffer (pH 8.0) 
            ♦ Guanidine thiocyanate
            ♦ Triton X100
            ♦ Tween 20
            ♦ 500 mM Sodium acetate (pH 5.2)
        Equipment and disposables
            ♦ Low DNA binding tube (e.g., Eppendorf LoBind tube, Cat.#0030108051) 

2.2 Preparation before DNA extraction 
       Silica solution
       To prepare 11 mL of silica solution
            ♦Wakosil (FUJIFILM [232-00841])    2.5 g
            ♦0.01 N HCl                                        11 mL
             *Before starting the DNA extraction, aliquot 10 µL of the solution into new low-bind tubes.
 
       SeraPure (Rohland et al. 2012)
       To prepare 50 mL of SeraPure
            ♦Sera-Mag SpeedBead Carboxylate-Modified [E3] Magnetic Particles      1 mL 
            ♦PEG8000                            9 g
            ♦NaCl                                    2.92 g
            ♦1 M Tris-HCl (pH 8.0)     500 μL 
            ♦500 mM EDTA (pH 8.0)  100 μL
            ♦50-mL tube
            ♦1.5-mL tube 
       1.     Take 1 mL of well-mixed Sera-Mag SpeedBeads in a 1.5-mL tube by inverting the tube.
       2.     Place the tube on a magnetic stand and wait for 5 min.
       3.     Remove the supernatant with a pipette tip and discard it.
       4.     Add 1 mL of TE buffer, remove the tube from the magnetic stand, and mix well by inverting.
       5.     Place the tube on the magnetic stand and wait for 5 min.
       6.     Remove the supernatant with a pipette tip and discard it.
       7.     Add 1 mL of TE buffer, remove the tube from the magnetic stand, and mix well by inverting.
       8.    Place the tube on the magnetic stand and wait for 5 minutes.
       9.     Remove the supernatant with a pipette tip and discard it.
       10.    Add 1 mL of TE buffer, remove the tube from the magnetic stand, and mix well by inverting.
       11.    In a 50-mL centrifuge tube, add 9 g of PEG8000 and 2.92 g of NaCl.
       12.    Add 500 μL of 1 M Tris-HCl (pH 8.0) and 100 μL of 500 mM EDTA (pH 8.0).
       13.    Fill up to 45 mL with sterile distilled water and completely dissolve the PEG.
       14.    Add 27.5 μL of Tween-20 and 1 mL of the solution from step 10.
       15.    Fill up to 50 mL with sterile distilled water.
 
       Binding Buffer
       To prepare 120 mL of binding buffer 
            ♦Guanidine thiocyanate                           70.9 g
            ♦Triton X100                                               4.8 g 
            ♦Tween 20                                                 12 %
            ♦500 mM Sodium acetate (pH 5.2)       240 μL
*If preparing this buffer is difficult, try using Buffer AL (QIAGEN, Cat. No.19075) as an alternative.
 
       Wash Buffer
       Stock wash buffer:ethanol = 1:4 ratio. (e.g., 20 mL stock wash buffer + 80 mL ethanol)
       Toprepare 50 mL of Stock wash buffer
            ♦500 mM EDTA (pH 8.0)      50 μL
            ♦5 M NaCl                               1 mL
            ♦1 M Tris-HCl (pH 8.0)       100 μL
 

Protocol materials

ReagentAL bufferQiagenCatalog #19075 

Sample storage using DESS solution (see Materials page)

To transport or store the DNA as undamaged as possible, place the specimen or environmental sample in DESS solution.

*Cautions when the sample is an animal
Since the DESS solution has low toxicity, animals are not immediately killed when placed in the DESS.
It is advisable to kill it somehow before putting the animals into the DESS.
This treatment is not particularly necessary for microscopic specimens (e.g. meiofauna).

Samples in DESS should be stored in the dark at room temperature (10 ~ 35゜C)

Add Amount10 µL   silica solution into new 1.5mL LoBind tube.

Note
Silica solution　*mix well before aliquote
  2.5g wakosil  (FUJIFILM, Cat.#232-00841)
  11mL 0.01N HCl 

Transfer AmountX µL   (eg. Amount500 µL   ) of DESS supernatant with the sample to the tube.

Add 1 volume of Binding buffer  (eg. Amount500 µL   ) to the tube.

Note
Binding Buffer 
  5M Guanidine Thiocyanate
  4% Triton X100
  10% Tween 20
  10mM Sodium acetate

*If it is not possible to prepare the Binding Buffer,  ReagentAL bufferQiagenCatalog #19075 can also be used.

Add 1 volume of Ethanol (eg.Amount500 µL   ) to the tube.

Mix by turning upside-down  for Duration00:05:00   at TemperatureRoom temperature   .

Centrifigation9100 x g, 20°C, 00:01:00   and discard the supernatant.

Add Amount500 µL   of Wash Buffer 1  and mix by vortexing.

Note
Wash Buffer
stock buffer : Ethanol = 1: 4 ratio

Stock buffer
 10mM TrisHCl pH8.0
 100 mM NaCl
 0.5mM EDTA pH8.0

Centrifigation9100 x g, 20°C, 00:01:00   and discard the supernatant with a pipette (or carefully with an aspirator).

Add Amount500 µL   of Wash Buffer 2  and mix by vortexing.

Centrifigation9100 x g, 20°C, 00:01:00   and discard the supernatant with a pipette (or carefully with an aspirator).

Centrifigation9100 x g, Room temperature, 00:01:00   and completely discard the supernatant with a pipette (or carefully with an aspirator).

Air dry for Duration00:01:00    at TemperatureRoom temperature  .

*Be careful not to over-dry.

Add Amount103 µL  TE and mix by vortexing.

Incubate at TemperatureRoom temperature   for Duration00:10:00   . (DNA dissolves in TE. )

*While  incubating, prepare the following
1) Prepare a new 1.5mL LoBind tube.
2) Add Amount10 µL   3M sodium acetate to the tube.
3) AddAmount1 µL   glycogen *optional

10m

Centrifigation9100 x g, Room temperature, 00:01:00  

Transfer the Amount100 µL  supernatant to a new 1.5 mL LoBind tube prepared in step 15.

AddAmount100 µL   isopropanol to the tube and mix by vortexing.

Temperature-20 °C   Duration00:05:00   ~

*Cool the centrifuge at this time.

Centrifigation20400 x g, 4°C  Duration00:30:00  and discard the supernatant with a pipette (or carefully with an aspirator).

*max speed

30m

AddAmount1 mL  75% Ethanol to the tube .
*Don't mix

Centrifigation20400 x g, 4°C  Duration00:01:00  and discard the supernatant with a pipette (or carefully with an aspirator).

*max speed

Centrifigation9100 x g, 00:01:00   and completely discard the supernatant with a pipette (or carefully with an aspirator).

*A small benchtop centrifuge can be used.

Air dry for Duration00:10:00  〜 Duration00:30:00    at TemperatureRoom temperature   in the dark.

*Be careful not to leave any ethanol residue and not to over-dry.

40m

AddAmount10 µL  0.1 TE to the tube  and mix by vortexing.

Incubate for  Duration00:10:00    at TemperatureRoom temperature   in the dark.

10m

PCR using Amount1 µL   supernatant.

Protocol references

1) DESS solution: https://www.youtube.com/watch?v=ye_1FRIR8bY)

Public workspaceNon-destructive DNA extraction from DESS preservation solution

Non-destructive DNA extraction from DESS preservation solution