Modified salting out method for high molecular weight gDNA extraction (oribatid mites)
Hüsna Öztoprak, Jens Bast
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
Steps
Version i) High-molecular gDNA extraction: DNA Extraction
Submerge cleansed specimen in 195µL 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.
Add 5µL proteinase K.
Vortex for 0h 0m 3s. Centrifuge briefly.
Incubate at 55°C for ~1h 0m 0s.
If there is indigestible debris left over centrifuge 18000rcf and transfer the supernatant to a fresh tube.
Add 1.5µL yeast tRNA, flick to mix briefly then spin down.
Add 65µL 5Molarity (M) NaCl and 290µL 96% EtOH, mix by inversion.
This should clarify the solution. Store at -20°C for 1h 0m 0s.
Version i) High-molecular gDNA extraction: DNA Purification
Optional : add 1µL Pellet Paint Co-Precipitant (for colorful pellet).
Spin down at 18000rcf,4°C.
Ghostly pellet should be visible.
Remove supernatant.
Add 0.5mL chilled 70% EtOH (make fresh).
Spin at 18000rcf.
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 21µL TE Buffer (elution buffer) and gently resuspend the DNA pellet with pipette mixing.
Let DNA resuspend at 4°C 1h 0m 0s.
Add 2µL RNase Cocktail. Incubate at 37°C for approx. 1h 0m 0s.
Version ii) High-molecular gDNA extraction preserving exoskeleton: DNA Extraction
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 195µL TNES buffer.
Add 5µL proteinase K.
Incubate at 37°C 1h 0m 0s.
Transfer specimen with sterile needle to a tube containing 70% EtOH and store it for morphological analysis.
Add 1.5µL yeast tRNA, flick to mix briefly then spin down.
Add 65µL 5Molarity (M) NaCl and 290µL 96% EtOH, mix by inversion.
This should clarify the solution. Store at -20°C for 1h 0m 0s.
Version iii) gDNA extraction with chitinase digestion: DNA Extraction
Submerge specimen in 195µL 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 2µL chitinase (1 U/ml).
Vortex for 0h 0m 5s. Centrifuge briefly.
Incubate at 55°C for ~1h 0m 0s.
Add 5µL proteinase K.
Vortex for 0h 0m 5s. Centrifuge briefly.
Incubate at 55°C for ~1h 0m 0s (completely dissolve the specimen).
If there is indigestible debris left over centrifuge 18000rcf and transfer the supernatant to a fresh tube.
Add 1.5µL yeast tRNA, flick to mix briefly then spin down.
Add 65µL 5Molarity (M) NaCl and 290µL 96% EtOH, mix by inversion.
This should clarify the solution. Store at -20°C for 1h 0m 0s.
