Modified salting out method for high molecular weight gDNA extraction (oribatid mites)

Hüsna Öztoprak; Jens Bast

Sep 26, 2023

Modified salting out method for high molecular weight gDNA extraction (oribatid mites)

DOI

dx.doi.org/10.17504/protocols.io.yxmvm3yybl3p/v1

Hüsna Öztoprak¹,
Jens Bast¹

¹Institute for Zoology, University of Cologne, Köln, Germany

h.oeztoprak

DOI: dx.doi.org/10.17504/protocols.io.yxmvm3yybl3p/v1

Protocol Citation: Hüsna Öztoprak, 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

Funders Acknowledgement:

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

111KB

Materials

Material and Regents

ReagentProteinase K, 2mLQiagenCatalog #19131 

ReagentYeast tRNA (10 mg/mL)Thermo Fisher ScientificCatalog #AM7119 

ReagentRNase Cocktail&trade; 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.: 
AB
NaCl400 mM
EDTA20 mM
Tris pH 8.050 mM
SDS0.5%

Version i) High-molecular gDNA extraction: DNA Extraction

2h 5m 3s

Submerge cleansed specimen in Amount195 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 Amount5 µL   proteinase K.

Vortex for Duration00:00:03  . Centrifuge briefly.

Incubate at Temperature55 °C   for ~Duration01:00:00  .
Note
 Completely dissolve the specimen.

If there is indigestible debris left over centrifuge Centrifigation18000 rcf, 00:05:00  and transfer the supernatant to a fresh tube.

Add Amount1.5 µL   yeast tRNA, flick to mix briefly then spin down.

Add Amount65 µL   Concentration5 Molarity (M)   NaCl and Amount290 µL   96% EtOH, mix by inversion.

This should clarify the solution. Store at Temperature-20 °C   for Duration01:00:00  .
Note
Solution can be stored DurationOvernight   at this step.

Version i) High-molecular gDNA extraction: DNA Purification

2h 20m

Optional: add Amount1 µL   Pellet Paint Co-Precipitant (for colorful pellet).

Spin down at Centrifigation18000 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 Amount0.5 mL   chilled 70% EtOH (make fresh).

Spin at Centrifigation18000 rcf, 00:05:00 .

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 Amount21 µL   TE Buffer (elution buffer) and gently resuspend the DNA pellet with pipette mixing.

Let DNA resuspend at Temperature4 °C   DurationOvernight  .

Add Amount2 µL   RNase Cocktail. Incubate at Temperature37 °C   for approx. Duration01:00:00  .
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).

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 Amount195 µL   TNES buffer.

Add Amount5 µL   proteinase K.

Incubate at Temperature37 °C   DurationOvernight  .

Transfer specimen with sterile needle to a tube containing 70% EtOH and store it for morphological analysis.

Add Amount1.5 µL   yeast tRNA, flick to mix briefly then spin down.

Add Amount65 µL   Concentration5 Molarity (M)   NaCl and Amount290 µL   96% EtOH, mix by inversion.

This should clarify the solution. Store at Temperature-20 °C   for Duration01:00:00  .
Note
DNA Purification follows the same procedure as above.

Version iii) gDNA extraction with chitinase digestion: DNA Extraction

3h 5m 10s

Submerge specimen in Amount195 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 Amount2 µL   chitinase (1 U/ml).

Vortex for Duration00:00:05  . Centrifuge briefly.

Incubate at Temperature55 °C   for ~Duration01:00:00  .

Add Amount5 µL   proteinase K.

Vortex for Duration00:00:05  . Centrifuge briefly.

Incubate at Temperature55 °C   for ~Duration01:00:00   (completely dissolve the specimen).

If there is indigestible debris left over centrifuge Centrifigation18000 rcf, 00:05:00  and transfer the supernatant to a fresh tube.

Add Amount1.5 µL   yeast tRNA, flick to mix briefly then spin down.

Add Amount65 µL   Concentration5 Molarity (M)   NaCl and Amount290 µL   96% EtOH, mix by inversion.

This should clarify the solution. Store at Temperature-20 °C   for Duration01:00:00  .
Note
DNA Purification follows the same procedure as above.

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

	A	B
	NaCl	400 mM
	EDTA	20 mM
	Tris pH 8.0	50 mM
	SDS	0.5%

Public workspaceModified salting out method for high molecular weight gDNA extraction (oribatid mites)

Modified salting out method for high molecular weight gDNA extraction (oribatid mites)