TITLE: Non-destructive DNA extraction from DESS preservation solution 
AUTHORS: Eri Ogiso-Tanaka, Minako Abe Ito, Daisuke Shimada

[DESCRIPTION]
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.

[STEPS]
1. 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)
3. 
Transfer X µL (eg. 500 µL ) of DESS supernatant with the sample to the tube.

4. Add 1 volume of Binding buffer  (eg. 500 µL ) to the tube.

 

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

6. Mix by turning upside-down  for 5 min at Room temperature .

2. Add 10 µL silica solution into new 1.5mL LoBind tube.

 

7. 9100 x g, 1 min, 20 °C  and discard the supernatant.


8. Add 500 µL of Wash Buffer 1  and mix by vortexing.

 

9. 9100 x g, 1 min, 20 °C  and discard the supernatant with a pipette (or carefully with an aspirator).
10. Add 500 µL of Wash Buffer 2  and mix by vortexing.
11. 9100 x g, 1 min, 20 °C  and discard the supernatant with a pipette (or carefully with an aspirator).
12. 9100 x g, 1 min, 20 Room temperature  and completely discard the supernatant with a pipette (or carefully with an aspirator).
13. Air dry for 1 min  at Room temperature.

*Be careful not to over-dry.


14. Add 103 µLTE and mix by vortexing.

15. Incubate at 55 Room temperature for 10 min . (DNA dissolves in TE. )

*While  incubating, prepare the following
1) Prepare a new 1.5mL LoBind tube.
2) Add 10 µL 3M sodium acetate to the tube.
3) Add1 µL glycogen *optional

16. 9100 x g, 1 min 
17. Transfer the 100 µLsupernatant to a new 1.5 mL LoBind tube prepared in step 15.

19. -20 °C 5 min ~

*Cool the centrifuge at this time.
20. 20400 x g, 4 °C 30 minand discard the supernatant with a pipette (or carefully with an aspirator).

*max speed

21. Add1 mL75% Ethanol to the tube .
*Don't mix
5. Add 1 volume of Ethanol (eg.500 µL ) to the tube.

18. Add100 µL isopropanol to the tube and mix by vortexing.
22. 20400 x g, 4 °C 1 minand discard the supernatant with a pipette (or carefully with an aspirator).

*max speed
23. 9100 x g, 1 min  and completely discard the supernatant with a pipette (or carefully with an aspirator).

*A small benchtop centrifuge can be used.
24. Air dry for 10 min〜 30 min  at Room temperature in the dark.

*Be careful not to leave any ethanol residue and not to over-dry.
25. Add10 µL0.1 TE to the tube  and mix by vortexing.
26. Incubate for  10 min  at Room temperature in the dark.


27. PCR using 1 µL supernatant.