TITLE: Modified salting out method for high molecular weight gDNA extraction (oribatid mites)
AUTHORS: Hüsna Öztoprak, Jens Bast

[DESCRIPTION]
This protocol describes a low-cost, high-molecular-weight genomic DNA extraction method for a single minuscule specimen (modified from Miller et al 1988). DNA extractions from oribatid mites are typically challenging, because of the small body size of 150-1400 µm. Their chitinous exoskeleton does not dissolve during DNA extraction, impedes DNA purification, and leads to additional loss of DNA. Therefore, high-molecular DNA from oribatid mites has been thus far unattainable, especially from single individuals. We established a high-molecular-weight gDNA extraction protocol for mites that enables the generation of high-quality phased genomes for small non-model organisms. There are three options to utilize this protocol: i) for high-molecular gDNA extraction ii) for high-molecular gDNA extraction, while preserving the exoskeleton for morphological analysis, and iii) DNA extraction with Chitinase to yield more gDNA. 

As specimens are collected from natural populations and are not cultured in the lab. They are cleansed prior to DNA extraction to minimize external contamination. Cleansing includes brushing the specimen in distilled water and in distilled water with detergent (fit GmbH, Zittau, Germany). Once the external residue is not visible anymore specimen is incubated in NaCIO 0.05% (DonKlorix; CP GABA GmbH, Hamburg, Germany) and ethanol 70% for 30 seconds each and rinsed in distilled water again.

[STEPS]
SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
1. Submerge cleansed specimen in 195 µL TNES buffer and flash freeze by holding tube in liquid nitrogen.
SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
2. Using a sterile pestle, homogenize by applying pressure to grind the specimen between pestle
head and the walls of the tube.
 

SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
3. Add 5 µL proteinase K.

SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
4. Vortex for 3 s. Centrifuge briefly.

SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
5. Incubate at 55 °C for ~60 min.
 

SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
5.1. If there is indigestible debris left over centrifuge 18000 rcf, 5 min and transfer the supernatant to a fresh tube.
SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
6. Add 1.5 µL yeast tRNA, flick to mix briefly then spin down.

SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
7. Add 65 µL 5 Molarity (M) NaCl and 290 µL 96% EtOH, mix by inversion.
SECTION: Version i) High-molecular gDNA extraction: DNA Extraction
8. This should clarify the solution. Store at -20 °C for 60 min.
 

SECTION: Version i) High-molecular gDNA extraction: DNA Purification
9. Optional: add 1 µL Pellet Paint Co-Precipitant (for colorful pellet).

SECTION: Version i) High-molecular gDNA extraction: DNA Purification
10. Spin down at 18000 rcf, 15 min, 4 °C.
 

SECTION: Version i) High-molecular gDNA extraction: DNA Purification
11. Ghostly pellet should be visible.
SECTION: Version i) High-molecular gDNA extraction: DNA Purification
12. Remove supernatant.
SECTION: Version i) High-molecular gDNA extraction: DNA Purification
13. Add 0.5 mL chilled 70% EtOH (make fresh).

SECTION: Version i) High-molecular gDNA extraction: DNA Purification
14. Spin at 18000 rcf, 5 min.

SECTION: Version i) High-molecular gDNA extraction: DNA Purification
15. Repeat ethanol rinse.
SECTION: Version i) High-molecular gDNA extraction: DNA Purification
16. Carefully remove supernatant.
SECTION: Version i) High-molecular gDNA extraction: DNA Purification
17. Leave tube open to air dry. Pellet should have a glassy appearance.
SECTION: Version i) High-molecular gDNA extraction: DNA Purification
18. Using a wide-bore pipette tip, add 21 µL TE Buffer (elution buffer) and gently resuspend the DNA pellet with pipette mixing.
SECTION: Version i) High-molecular gDNA extraction: DNA Purification
19. Let DNA resuspend at 4 °C 60 min.

SECTION: Version i) High-molecular gDNA extraction: DNA Purification
20. Add 2 µL RNase Cocktail. Incubate at 37 °C for approx. 60 min.
 

SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
21. To observe the specimen under a microscope, cleansed specimens are placed on a sterile slide and submerged with TNES buffer until fully covered.
SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
22. Remove one genital plate cautiously with a sharp needle and stir tissue without destroying the
exoskeleton.
SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
23. Transfer specimen in 195 µL TNES buffer.

SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
24. Add 5 µL proteinase K.

SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
25. Incubate at 37 °C 60 min.

SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
26. Transfer specimen with sterile needle to a tube containing 70% EtOH and store it for morphological analysis.
SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
27. Add 1.5 µL yeast tRNA, flick to mix briefly then spin down.

SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
28. Add 65 µL 5 Molarity (M) NaCl and 290 µL 96% EtOH, mix by inversion.

SECTION: Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
29. This should clarify the solution. Store at -20 °C for 60 min.
 

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
30. Submerge specimen in 195 µL TNES buffer and flash freeze by holding tube in liquid N.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
31. Using a sterile pestle, homogenize by applying pressure to grind the specimen between pestle head and the walls of the tube.
SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
32. Add 2 µL chitinase (1 U/ml).

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
33. Vortex for 5 s. Centrifuge briefly.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
34. Incubate at 55 °C for ~60 min.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
35. Add 5 µL proteinase K.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
36. Vortex for 5 s. Centrifuge briefly.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
37. Incubate at 55 °C for ~60 min (completely dissolve the specimen).
SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
37.1. If there is indigestible debris left over centrifuge 18000 rcf, 5 min and transfer the supernatant to a fresh tube.
SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
38. Add 1.5 µL yeast tRNA, flick to mix briefly then spin down.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
39. Add 65 µL 5 Molarity (M) NaCl and 290 µL 96% EtOH, mix by inversion.

SECTION: Version iii) gDNA extraction with chitinase digestion: DNA Extraction
40. This should clarify the solution. Store at -20 °C for 60 min.