High-throughput papain-based DNA extraction from whole invertebrates

Jordan P Cuff, James JN Kitson, Rosa Whittingham

Published: 2022-08-17 DOI: 10.17504/protocols.io.261genb8dg47/v1

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.

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.

Steps

DNA extraction: purification

0.1.

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

0.2.

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	42.2 mL
Ethanol (100 %)	220 μL	42.2 μL

Collection of samples

Consider how systematic the study needs to be and the various constraints imposed on the data by the study design.

Add 200 μL of the supernatant from STEP 10 into each well containing Protein Denaturation Buffer and ethanol.

Add all of the sample solution (~ 600 μL) to a well in a 96-well DNA membrane spin column 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).

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.

Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.

Note

If the centrifuge cannot reach 6000 x g , a longer centrifugation (e.g., 10 minutes) will work.

Store samples at -20 °C until ready to process.

Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.

Preparation and homogenisation of samples

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.

4.1.

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

Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.

Note

If the centrifuge cannot reach 6000 x g , a longer centrifugation (e.g., 5 minutes) will work.

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

Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.

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.

6.1.

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

Centrifuge at 20,000 x g for 3 minutes and discard the flow-through.

Note

If the centrifuge cannot reach 20,000 x g , a longer centrifugation (e.g., 15 minutes) will work.

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.

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.

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 NaHCO₃into 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).

8.1.

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

8.2.

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

Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.

Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours). Alternatively, incubate at 56°C for three hours.

9.1.

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

10.

Centrifuge at ≥ 6000 x g for 1 minute. The DNA is now in the collection plate and can be taken forward to amplification.

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.

Sample washes and DNA elution

10.

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.

10.1.

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

10.2.

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

11.

Vortex samples briefly to ensure thorough mixing

12.

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).

13.

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.

14.

Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.

14.1.

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

15.

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.

16.

Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.

16.1.

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

17.

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.

18.

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.

19.

Add 100 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.

19.1.

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

20.

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

High-throughput papain-based DNA extraction from whole invertebrates

Abstract

Before start

Steps

DNA extraction: purification

Collection of samples

Preparation and homogenisation of samples

Sample washes and DNA elution

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