TITLE: High-throughput papain-based DNA extraction from whole invertebrates
AUTHORS: Rosa Whittingham, James JN Kitson, Jordan P Cuff

[DESCRIPTION]
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]
SECTION: Collection of samples
1. Consider how systematic the study needs to be and the various constraints imposed on the data by the study design. 
SECTION: DNA extraction: purification
0.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction: purification
1. Add 200 μL of the supernatant from STEP 10 into each well containing Protein Denaturation Buffer and ethanol. 
SECTION: DNA extraction: purification
0.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
2. 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. 
 

SECTION: DNA extraction: purification
3. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
4. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
5. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
4.1. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction: purification
6. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
6.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction: purification
7. Centrifuge at 20,000 x g for 3 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
8. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
9. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction: purification
9.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

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

SECTION: DNA extraction: purification
None. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed below) to each well of the 2.2 ml 96-well plate.
SECTION: DNA extraction: purification
None. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction: purification
None. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin column and cover with a breathable seal. 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 10 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 5 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
None. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 15 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction: purification
None. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 2 minutes. The DNA is now in the collection plate and can be taken forward to amplification. 
 

SECTION: Initial digestion of OPM larvae
None. Grind the larvae in a Geno/Grinder 2010 at full speed (1750 RPM) for 4 minutes.
 

SECTION: Initial digestion of OPM larvae
None. Centrifuge at 4,000 g for 2 minutes.
SECTION: Initial digestion of OPM larvae
None. Remove the ball bearings using a magnet, taking care not to cross-contaminate the samples.
SECTION: Initial digestion of OPM larvae
None. To the lysate, add 1 mL of freshly-prepared Proteinase Buffer master mix of Lysis Buffer 2 and proteinase K (detailed in the sub-steps below) and vortex to mix.
SECTION: Initial digestion of OPM larvae
None. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Initial digestion of OPM larvae
None. Per sample, the Proteinase Buffer master mix should comprise:

 

SECTION: Initial digestion of OPM larvae
None. Incubate at 37 °C overnight (12-16 hours)
 

SECTION: Initial digestion of OPM larvae
None. Centrifuge at 4000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
None. Transfer 1 mL of the supernatant to a clean 2.2 mL deep-well 96-well plate for archiving/backup and retain the rest in the original plate.
SECTION: Initial digestion of OPM larvae
None. Centrifuge at 2000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
None. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: Preparation and homogenisation of samples
5. Add individual invertebrates to each well of the plate.


 

SECTION: Collection of samples
2. Collect individual invertebrates from the field into a suitable preservative (e.g., 100 % ethanol).
 

SECTION: Collection of samples
3. Store samples at -20 °C until ready to process.
SECTION: Preparation and homogenisation of samples
7. Directly from the freezer, grind the bulk samples in a Geno/Grinder at full speed (1750 RPM) for 2 minutes.
 

SECTION: Preparation and homogenisation of samples
6. Freeze the samples overnight at -20 °C.
 

SECTION: Preparation and homogenisation of samples
9. Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours).  Alternatively, incubate at 56°C for three hours.
SECTION: Preparation and homogenisation of samples
8. 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:

 


 

SECTION: Preparation and homogenisation of samples
8.1. Lysis Buffer 1 should be pH 9 and comprised of the following reagents:
 

SECTION: Preparation and homogenisation of samples
8.2. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 

SECTION: 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.
SECTION: Sample washes and DNA elution
13. Centrifuge at ≥ 6,000 x g for 10 minutes and discard the flow-through.
 

SECTION: Sample washes and DNA elution
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. 
 

SECTION: Sample washes and DNA elution
18. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: Sample washes and DNA elution
14. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: Sample washes and DNA elution
15. Centrifuge at ≥ 6,000 x g for 5 minutes and discard the flow-through.
 

SECTION: Sample washes and DNA elution
14.1. Wash Buffer 1 should be comprised of the following reagents: 
 

SECTION: Sample washes and DNA elution
16. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: Sample washes and DNA elution
17. Centrifuge at ≥ 6,000 x g for 15 minutes and discard the flow-through.
 

SECTION: Sample washes and DNA elution
16.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:
 

SECTION: Sample washes and DNA elution
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.
SECTION: Sample washes and DNA elution
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. 
 

SECTION: Sample washes and DNA elution
19.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 

SECTION: Preparation and homogenisation of samples
4. Add one 3 mm hardened carbon steel bead to each well of a 1.2 mL 96-well plate.
 

SECTION: Sample washes and DNA elution
11. Vortex samples briefly to ensure thorough mixing
SECTION: Sample washes and DNA elution
10.1. Protein Denaturation Buffer should be comprised of the following reagents:
     ABCD1ReagentRequired concentration in bufferChemical molarityAmount per 100 mL2Guanidine HCl5 M95.5347.7 g3H2O--100 mL

SECTION: Sample washes and DNA elution
10.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:
    ABC1ReagentAmount per sampleAmount per 96-well plate2Protein Denaturation Buffer220 μL21.1 mL3Ethanol (100 %)220 μL21.1 μL