Sep 26, 2023
Modified salting out method for high molecular weight gDNA extraction (oribatid mites)
- Hüsna ztoprak1,
- Jens Bast1
- 1Institute for Zoology, University of Cologne, Köln, Germany
Protocol Citation: Hüsna ztoprak, Jens Bast 2023. Modified salting out method for high molecular weight gDNA extraction (oribatid mites). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3yybl3p/v1
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. It enables the generation of HMW gDNA of ~20 ng, even up to 200 ng of a single oribatid mite, depending on the genus.
It also works for collembolans and nematodes.
Created: September 25, 2023
Last Modified: September 26, 2023
Protocol Integer ID: 88360
Keywords: dna extractions from oribatid mite, weight gdna extraction protocol for mite, method for high molecular weight gdna extraction, molecular dna from oribatid mite, high molecular weight gdna extraction, weight gdna extraction protocol, dna extraction with chitinase, weight genomic dna extraction method, molecular gdna extraction, molecular gdna extraction ii, dna extraction, dna purification, oribatid mite, more gdna, genomic dna, mite, dna, additional loss of dna, extraction, molecular dna, chitinase, single minuscule specimen, phased genome, chitinous exoskeleton
Funders Acknowledgements:
German Research Foundation Emmy Noether
Grant ID: grant BA 5800/4-1
Abstract
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.
Attachments
nn7eb8kcf.pdf
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Materials
Material and Regents
Proteinase K, 2mLQiagenCatalog #19131
Yeast tRNA (10 mg/mL)Thermo Fisher ScientificCatalog #AM7119
RNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
Solutions
1. TNES buffer (see Recipes)
Recipes
1. Final concentration of TNES buffer freshly made before each extraction, ddH20 used to dilute.:
| A | B | |
| NaCl | 400 mM | |
| EDTA | 20 mM | |
| Tris pH 8.0 | 50 mM | |
| SDS | 0.5% |
Troubleshooting
Version i) High-molecular gDNA extraction: DNA Extraction
2h 5m 3s
Submerge cleansed specimen in 195 µL TNES buffer and flash freeze by holding tube in liquid nitrogen.
Using a sterile pestle, homogenize by applying pressure to grind the specimen between pestle
head and the walls of the tube.
Note
If low DNA yield is expected. Leave pestle in tube to ensure maximum digestion of material.
Consider including multiple (3x) freeze and thaw cycles and vortexing to disrupt tissue fully.
Add 5 µL proteinase K.
Vortex for 00:00:03 . Centrifuge briefly.
3s
Incubate at 55 °C for ~01:00:00 .
Note
Completely dissolve the specimen.
1h
If there is indigestible debris left over centrifuge 18000 rcf, 00:05:00 and transfer the supernatant to a fresh tube.
5m
Add 1.5 µL yeast tRNA, flick to mix briefly then spin down.
Add 65 µL 5 Molarity (M) NaCl and 290 µL 96% EtOH, mix by inversion.
This should clarify the solution. Store at -20 °C for 01:00:00 .
Note
Solution can be stored Overnight at this step.
1h
Version i) High-molecular gDNA extraction: DNA Purification
2h 20m
Optional: add 1 µL Pellet Paint Co-Precipitant (for colorful pellet).
Spin down at 18000 rcf, 4°C, 00:15:00 .
Note
Know the expected position of the DNA pellet, as it can be difficult to see.
15m
Ghostly pellet should be visible.
Remove supernatant.
Add 0.5 mL chilled 70% EtOH (make fresh).
Spin at 18000 rcf, 00:05:00 .
5m
Repeat ethanol rinse.
Carefully remove supernatant.
Leave tube open to air dry. Pellet should have a glassy appearance.
Using a wide-bore pipette tip, add 21 µL TE Buffer (elution buffer) and gently resuspend the DNA pellet with pipette mixing.
Let DNA resuspend at 4 °C Overnight .
1h
Add 2 µL RNase Cocktail. Incubate at 37 °C for approx. 01:00:00 .
Note
Note: For femto pulse systems elution in TE Buf er, more specifically EDTA is not recommended. Alternatively use 0.1 mM EDTA or EB, Tris-HCl (pH 8-8.5).
1h
Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
2h
To observe the specimen under a microscope, cleansed specimens are placed on a sterile slide and submerged with TNES buffer until fully covered.
Remove one genital plate cautiously with a sharp needle and stir tissue without destroying the
exoskeleton.
Transfer specimen in 195 µL TNES buffer.
Add 5 µL proteinase K.
Incubate at 37 °C Overnight .
1h
Transfer specimen with sterile needle to a tube containing 70% EtOH and store it for morphological analysis.
Add 1.5 µL yeast tRNA, flick to mix briefly then spin down.
Add 65 µL 5 Molarity (M) NaCl and 290 µL 96% EtOH, mix by inversion.
This should clarify the solution. Store at -20 °C for 01:00:00 .
Note
DNA Purification follows the same procedure as above.
1h
Version iii) gDNA extraction with chitinase digestion: DNA Extraction
3h 5m 10s
Submerge specimen in 195 µL TNES buffer and flash freeze by holding tube in liquid N.
Using a sterile pestle, homogenize by applying pressure to grind the specimen between pestle head and the walls of the tube.
Add 2 µL chitinase (1 U/ml).
Vortex for 00:00:05 . Centrifuge briefly.
5s
Incubate at 55 °C for ~01:00:00 .
1h
Add 5 µL proteinase K.
Vortex for 00:00:05 . Centrifuge briefly.
5s
Incubate at 55 °C for ~01:00:00 (completely dissolve the specimen).
1h
If there is indigestible debris left over centrifuge 18000 rcf, 00:05:00 and transfer the supernatant to a fresh tube.
5m
Add 1.5 µL yeast tRNA, flick to mix briefly then spin down.
Add 65 µL 5 Molarity (M) NaCl and 290 µL 96% EtOH, mix by inversion.
This should clarify the solution. Store at -20 °C for 01:00:00 .
Note
DNA Purification follows the same procedure as above.
1h
Protocol references
References
Laumer, C. 2023. “Picogram input multimodal sequencing (PiMmS).” protocols.io
https://dx.doi.org/10.17504/protocols.io.rm7vzywy5lx1/v1
Miller, S. A., D. D. Dykes, and H. F. Polesky. 1988. “A Simple Salting out Procedure for Extracting DNA from Human Nucleated Cells.” Nucleic Acids Research 16 (3): 1215