TITLE: High-throughput and cost effective pan trap DNA extraction
AUTHORS: Jordan P Cuff, James JN Kitson

[DESCRIPTION]
This protocol is designed for extracting DNA from pan trap-collected invertebrates for biomonitoring and community metabarcoding. The reagents and methods proposed offer a cost effective and high-throughput method for molecular diversity analyses of bulk samples using standard lab equipment.

[STEPS]
SECTION: Preparation and homogenisation of samples
15. Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours).
SECTION: DNA extraction and purification
20. 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: DNA extraction and purification
21. Centrifuge at ≥ 6,000 x g for 10 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
22. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction and purification
22.1. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction and purification
23. Centrifuge at ≥ 6,000 x g for 5 minutes and discard the flow-through.
 

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

 

SECTION: DNA extraction and purification
25. Centrifuge at ≥ 6,000 x g for 15 minutes and discard the flow-through.
 

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

SECTION: DNA extraction and purification
27. 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 and purification
27.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction and purification
28. 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: Collection of pan trap samples
1. Select suitable sites and locations for pan traps. Consider how systematic the study needs to be and the various constraints imposed on the data by the study design.
SECTION: Preparation and homogenisation of samples
14. To the 1 mL lysate, add 500 μL of freshly-prepared Proteinase Buffer, a master mix of Lysis Buffer 2 and proteinase K (detailed in the sub-steps below) and vortex to mix.
SECTION: Preparation and homogenisation of samples
14.1. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Preparation and homogenisation of samples
14.2. Per plate, the Proteinase Buffer master mix should comprise:

 

SECTION: Preparation and homogenisation of samples
16. Centrifuge at 4,000 x g for 4 minutes.
SECTION: Preparation and homogenisation of samples
17. Transfer 1 mL of the supernatant to a clean 2.2 mL deep-well 96-well plate for archiving/backup.
SECTION: Preparation and homogenisation of samples
18. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: DNA extraction and purification
19. 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.
SECTION: DNA extraction and purification
19.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction and purification
19.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction: purification
None. Add 200 μL of the supernatant from STEP 10 into each well containing Protein Denaturation Buffer and ethanol. 
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 DNA membrane spin column and cover with a breathable seal. 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute 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. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Wash Buffer 1 should be comprised of the following reagents:

 

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. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Centrifuge at 20,000 x g for 3 minutes and discard the flow-through.
 

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. 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 1 minute. The DNA is now in the collection plate and can be taken forward to amplification. 
 

SECTION: Initial digestion of OPM larvae
None. When ready to begin tissue digestion, defrost the tubes containing dead larvae in Lysis Buffer 1.
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
6. Add three 3 mm hardened carbon steel beads to the tube.
 

SECTION: Preparation and homogenisation of samples
9. Two lysis options are presented below. One standard protocol using Proteinase K to lyse a subsample of each trap (this may reduce detectable diversity but increases efficiency), and the other uses papain (a cheap alternative to Proteinase K) to lyse whole samples.
SECTION: Collection of pan trap samples
2. Set a pan trap at approximately the same height as the surrounding vegetation. 
 

SECTION: Collection of pan trap samples
3. Fill the traps with water containing detergent to reduce surface tension. Leave the traps out for 72 hours.
 

SECTION: Collection of pan trap samples
4. Pour the trap contents through a sterilised funnel into a suitably sized pot labelled with all necessary sample information for transport to the laboratory. Store samples at -20 °C until ready to process.
SECTION: Preparation and homogenisation of samples
8. Directly from the freezer, grind the bulk samples in a Geno/Grinder at full speed (1750 RPM) for 4 minutes.
 

SECTION: Preparation and homogenisation of samples
13. Take a subsample of 1 mL lysate from the supernatant and transfer it to a 2.2 mL 96-well plate. Store the remaining lysate and sample from the falcon tube at -20 °C.
 

SECTION: Preparation and homogenisation of samples
11. Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours).
SECTION: Preparation and homogenisation of samples
12. Vortex to mix and centrifuge at 4,000 x g for 4 minutes.
SECTION: Preparation and homogenisation of samples
12. Centrifuge at 4,000 x g for 2 minutes.
SECTION: Preparation and homogenisation of samples
13. Transfer 1 mL of the supernatant to a clean 2.2 mL deep-well 96-well plate for archiving/backup.
SECTION: DNA extraction and purification
15. 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.
SECTION: Preparation and homogenisation of samples
14. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: DNA extraction and purification
16. 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: DNA extraction and purification
15.1. Protein Denaturation Buffer should be comprised of the following reagents:
 
 

SECTION: DNA extraction and purification
15.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:
 
 

SECTION: DNA extraction and purification
17. Centrifuge at ≥ 6,000 x g for 10 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
18. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction and purification
18.1. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction and purification
19. Centrifuge at ≥ 6,000 x g for 5 minutes and discard the flow-through.
 

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

 

SECTION: DNA extraction and purification
21. Centrifuge at ≥ 6,000 x g for 15 minutes and discard the flow-through.
 

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

SECTION: DNA extraction and purification
23. 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 and purification
23.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction and purification
24. 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: Preparation and homogenisation of samples
5. Wash samples with water in a fine mesh sieve to remove external contaminants, and transfer to a 50 mL falcon tube.
SECTION: Preparation and homogenisation of samples
7. Freeze the samples overnight at -80 °C.
SECTION: Preparation and homogenisation of samples
10. Add an appropriate volume of fresh Lysis Buffer 1 (detailed in the sub-step below) to sufficiently cover the specimens.
 

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

 

SECTION: Preparation and homogenisation of samples
10. To each sample, add 30 mL of fresh Lysis Buffer 1 and 15 mL of Proteinase Buffer (a master mix of Lysis Buffer 2 and papain; all reagents detailed in the sub-steps below).
 

 

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

 

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

 

SECTION: Preparation and homogenisation of samples
10.3. Per sample, the Proteinase Buffer master mix should comprise:
 
 

SECTION: Preparation and homogenisation of samples
11. Grind the samples in a Geno/Grinder at full speed (1750 RPM) for 2 minutes.