Aug 16, 2022
High-throughput papain-based DNA extraction from whole invertebrates
- 1University of York;
- 2Newcastle University
Protocol Citation: Rosa Whittingham, James JN Kitson, Jordan P Cuff 2022. High-throughput papain-based DNA extraction from whole invertebrates. protocols.io https://dx.doi.org/10.17504/protocols.io.261genb8dg47/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
Created: July 13, 2022
Last Modified: August 16, 2022
Protocol Integer ID: 66606
Keywords: metabarcoding, biomonitoring, entomology, high-throughput sequencing, community ecology, field techniques, parasitism, dietary analysis, barcoding
Abstract
This protocol is designed for extracting DNA from individual invertebrates in 96-well plate format for downstream barcoding or metabarcoding. The reagents and methods proposed offer a cost effective and high-throughput method for molecular identification, dietary analysis, parasitism diagnostics and more from individual invertebrate samples using standard lab equipment.
Image Attribution
Biorender.com
Materials
For field collection and initial storage:
- Small collection tubes for storage of samples (e.g., 1.5 mL microcentifuge tubes)
- 100 % ethanol
For DNA extraction:
- Hardened 3 mm carbon steel ball bearings
- 1.2 mL 96-well plates (or larger) for initial lysis and protein denaturation
- 2.2 mL deep well plates for spin-column flow-through (these can be bleached and reused across sessions)
- Silica membrane 96-well spin-column plates
- 0.5 mL deep-well 96-well plates to collect eluted DNA
- Breathable plate seals
- Plate seals for long-term storage
Buffers and reagents:
- Sodium chloride
- 1 M Tris-HCl
- 0.5 M EDTA
- Nuclease-free water
- SDS
- Guanidine HCl
- 100 % ethanol
- Papain (100 mg/mL)
Equipment:
- -20 °C freezer
- Geno/Grinder 2010 or similar bead beater for homogenisation
Safety warnings
Check safety guidelines for individual reagents before commencing work. Some reagents will be toxic, corrosive or will otherwise present health and safety risks. Appropriate personal protective equipment should be used at all times, not only for personal safety but also reduction of contamination risk.
Before start
Consider steps that can be taken to limit cross-contamination throughout the process. Contamination between samples and from the environment can produce false positive data and inaccurate results to which downstream analyses like DNA metabarcoding can be incredibly sensitive.
Collection of samples
Collection of samples
Consider how systematic the study needs to be and the various constraints imposed on the data by the study design.
Collect individual invertebrates from the field into a suitable preservative (e.g., 100 % ethanol).
Note
Ensure that the collection equipment is clean and free of DNA contaminants by cleaning it with Chemgene or diluted bleach prior to and between each use.
Ensure appropriate permissions are in place for the site. If your site is publicly accessible or likely to be visited, consider using signage to reduce the risk of tampering (e.g., vandalism or benevolent liberation of the dead invertebrates).
Consider as well the ethical implications of your collections. Limit unnecessary collection and implement measures to limit bycatch of non-target organisms where applicable.
Store samples at -20 °C until ready to process.
Preparation and homogenisation of samples
Preparation and homogenisation of samples
1d 8h 28m
1d 8h 28m
Add one 3 mm hardened carbon steel bead to each well of a 1.2 mL 96-well plate.
Note
Beads are usually shipped coated in manufacturing oil (especially the carbon steel beads). To remove this, place beads in a borosilicate glass beaker or Duran bottle with plastic pouring lip and lid removed then bake for at least 12 hours at 250 °C.
96-well bead dispensers make this step significantly quicker and less liable to contamination.
5m
Add individual invertebrates to each well of the plate.
Note
Ensure appropriate sterilisation of tweezers between each transfer of samples by using bleach, ethanol and water to prevent cross contamination
15m
Freeze the samples overnight at -20 °C.
Note
This will ensure the specimens are brittle and grind adequately; -80 °C will facilitate even better grinding, but it will make the plastic brittle too, so check the plasticware will survive without samples before proceeding.
16h
Directly from the freezer, grind the bulk samples in a Geno/Grinder at full speed (1750 RPM) for 2 minutes.
Note
If the sample is not fully homogenised, repeat this step.
2m
To each sample, add 140 μL of fresh Lysis Buffer 1, 60 μL of Lysis Buffer 2 and 20 μL of 100 mg/mL papain:
Note
Borosilicate bottles are ideal for making up larger quantities of buffer.
To sterilise borosilicate bottles prior to making up the buffers, acid washing with ~100 mL 0.4 M hydrochloric acid followed by neutralisation with ~100 mL 0.4 M sodium bicarbonate is ideal for sterilisation without introducing bleach or other chemicals that might destroy or contaminate the DNA. Following neutralisation, wash twice with ~100 mL water to remove the reagents. Residual amounts of NaCl may remain, but this is inert and will be present in many of the buffers anyway.
For 0.4 M sodium bicarbonate add 33.604 g NaHCO3 into 1000 mL water.
Note
Proteinase K can be used instead of papain, but is much more expensive. Given the low cost of papain, it is used at ten-fold concentration to ensure adequate lysis, but could be used as per the standard concentration of Proteinase K (10 mg/mL).
2m
Lysis Buffer 1 should be pH 9 and comprised of the following reagents:
| A | B | C | D | |
| Reagent | Required concentration in buffer | Chemical molarity | Amount per 100 mL | |
| NaCl | 120 mM | 58.44 | 0.701 g | |
| 1 M Tris-Hcl | 50 mM | - | 5 mL | |
| 0.5 M EDTA | 20 mM | - | 4 mL | |
| Water | - | - | 91 mL |
2m
Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
| A | B | C | D | |
| Reagent | Required concentration in buffer | Chemical molarity | Amount per 100 mL | |
| Sodium chloride | 120 mM | 58.44 | 0.701 g | |
| 1 M Tris-Hcl | 50 mM | - | 5 mL | |
| 0.5 M EDTA | 20 mM | - | 4 mL | |
| SDS | 3 % | - | 3 g | |
| H2O | - | - | 91 mL |
2m
Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours). Alternatively, incubate at 56°C for three hours.
16h
Sample washes and DNA elution
Sample washes and DNA elution
1h 6m
1h 6m
Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed in the sub-step below) to each well of the 2.2 mL 96-well plate.
Create master mix of 1:1 ratio of protein denaturation buffer and ethanol.
2m
Protein Denaturation Buffer should be comprised of the following reagents:
| A | B | C | D | |
| Reagent | Required concentration in buffer | Chemical molarity | Amount per 100 mL | |
| Guanidine HCl | 5 M | 95.53 | 47.7 g | |
| H2O | - | - | 100 mL |
2m
Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:
| A | B | C | |
| Reagent | Amount per sample | Amount per 96-well plate | |
| Protein Denaturation Buffer | 220 μL | 21.1 mL | |
| Ethanol (100 %) | 220 μL | 21.1 μL |
2m
Vortex samples briefly to ensure thorough mixing
1m
Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin-column plate and cover with a breathable seal.
Note
Ensure there is a suitable reservoir beneath into which the flow-through will go (e.g., 2.2 mL deep-well plate).
5m
Centrifuge at ≥ 6,000 x g for 10 minutes and discard the flow-through.
Note
If the centrifuge cannot reach 6000 x g, a longer centrifugation will work.
10m
Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
2m
Wash Buffer 1 should be comprised of the following reagents:
| A | B | C | D | |
| Reagent | Required concentration in buffer | Chemical molarity | Amount per 100 mL | |
| Guanidine HCl | 7 M | 95.53 | 29.4 g | |
| Ethanol | 56 % | - | 56 mL | |
| H2O | - | - | 44 mL |
2m
Centrifuge at ≥ 6,000 x g for 5 minutes and discard the flow-through.
Note
If the centrifuge cannot reach 6000 x g, a longer centrifugation will work.
5m
Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
2m
Wash Buffer 2 should be pH ~7 and comprised of the following reagents:
| A | B | C | D | |
| Reagent | Required concentration in buffer | Chemical molarity | Amount per 100 mL | |
| Ethanol (100 %) | 70 % | 58.44 | 70 mL | |
| 1 M Tris-Hcl | 10 mM | - | 1 mL | |
| H2O | - | - | 29 mL |
2m
Centrifuge at ≥ 6,000 x g for 15 minutes and discard the flow-through.
Note
If the centrifuge cannot reach 6,000 x g, a longer centrifugation will work.
15m
Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
Note
The liquid level following the final wash will be close to the base of the spin column, so take care not to let it touch to prevent ethanol transfer to the soon-to-be eluted DNA.
1m
Add 100 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
6m
Elution Buffer should be pH ~7 and comprised of the following reagents:
| A | B | C | D | |
| Reagent | Required concentration in buffer | Chemical molarity | Amount per 100 mL | |
| 1 M Tris-Hcl | 10 mM | - | 1 mL | |
| H2O | - | - | 99 mL |
2m
Centrifuge at ≥ 6000 x g for 2 minutes. The DNA is now in the collection plate and can be taken forward to molecular analysis.
Note
Steps 20-21 can be repeated for increased DNA yield but a lower overall concentration.
If the centrifuge cannot reach 6000 x g, a longer centrifugation (e.g., 5 minutes) will work, although should not be necessary for this step.
2m