TITLE: Tissue extraction from whole caterpillars
AUTHORS: James JN Kitson

[DESCRIPTION]
This protocol is designed for extracting DNA from Lepidopteran larvae but it will work on most animal tissue with some modifications to tube volumes and homogenisation settings.


[STEPS]
SECTION: Initial digestion of OPM larvae
8. The next steps depend on the intended throughput: either single sample spin-columns or 96-well spin-column plates.
SECTION: Initial digestion of OPM larvae
13. Incubate at 37 °C overnight (12-16 hours)

37 °C 
SECTION: Initial digestion of OPM larvae
11. Centrifuge at 4,000 x g for 2 min.
SECTION: DNA extraction: purification
19. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin column (we use SD5005 from NBS Biologicals) 
 

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


SECTION: DNA extraction: purification
21. Add 500 μL Wash Buffer 1 to each spin column.
SECTION: DNA extraction: purification
21.1. Wash Buffer 1 should be comprised of the following reagents:

 

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


SECTION: DNA extraction: purification
23. Add 500 μL Wash Buffer 2 to each spin column.
SECTION: DNA extraction: purification
23.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction: purification
24. Centrifuge at 20,000 x g for 3 minutes.


SECTION: DNA extraction: purification
25. Discard the collection tube and replace it with a new 1.5 mL microcentrifuge tube.
SECTION: DNA extraction: purification
26. Add 100 - 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes.
SECTION: DNA extraction: purification
26.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

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

SECTION: Collection of larvae
6. Repeat until a sufficient sample size has been collected.
SECTION: Collection of larvae
7. Place all sample tubes from a session in a zip-lock bag and keep cold under dry ice or place in a portable freezer to freeze for transport.
SECTION: Initial digestion of OPM larvae
10. Grind the larvae is a tissue homogeniser until homogenised.

 

SECTION: Initial digestion of OPM larvae
12. To the lysate, add 1 mL 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: Initial digestion of OPM larvae
12.1. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Initial digestion of OPM larvae
12.2. Per sample, the Proteinase Buffer master mix should comprise:

 

SECTION: Initial digestion of OPM larvae
14. Centrifuge at 4000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
15. Transfer 1.5 mL of the supernatant to a clean 2 mL screw-cap tube for archiving/backup.
SECTION: Initial digestion of OPM larvae
16. Centrifuge at 10,000 x g for 1 minute.
SECTION: Initial digestion of OPM larvae
17. Transfer 200 μL of the supernatant to a clean 1.5 mL microcentrifuge tube. 
 

SECTION: DNA extraction: purification
18. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed below) to each sample.
SECTION: DNA extraction: purification
18.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

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

 

SECTION: DNA extraction: purification
None. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed below) to each well of a new 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. 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
9. When ready to begin tissue digestion, defrost the tubes containing dead larvae in Lysis Buffer 1.
SECTION: Initial digestion of OPM larvae
9. When ready to begin tissue digestion, defrost the tubes containing dead larvae in Lysis Buffer 1.
SECTION: DNA extraction: 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: purification
19.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

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

 

SECTION: DNA extraction: purification
20. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin column (we use SD5007 from NBS Biologicals) and cover with a breathable seal. 
 

SECTION: DNA extraction: purification
21. Centrifuge at maximum speed for 10 minutes and discard the flow-through.
 

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

 

SECTION: DNA extraction: purification
23. Centrifuge at maximum speed for 5 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
24. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
26. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
24.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
25. Centrifuge at maximum speed for 15 minutes and discard the flow-through.
 

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

SECTION: DNA extraction: purification
28. 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
10. Grind the larvae is a tissue homogeniser until homogenised.

 

SECTION: Initial digestion of OPM larvae
11. Centrifuge at 4,000 x g for 2 minutes.
SECTION: Initial digestion of OPM larvae
12. To the lysate, add 1 mL 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: Initial digestion of OPM larvae
12.1. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Initial digestion of OPM larvae
12.2. Per plate, the Proteinase Buffer master mix should comprise:

 

SECTION: Initial digestion of OPM larvae
13. Incubate at 37 °C overnight (12-16 hours)

37 °C 

SECTION: Initial digestion of OPM larvae
14. Centrifuge at 4000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
15. 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
16. Centrifuge at 2000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
17. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: Collection of larvae
5. Using sterile disposable forceps, place individual caterpilars into the pre-prepared collection tubes containing the hardened steel ball bearings and Lysis Buffer 1. 
 

SECTION: Collection of larvae
3. Prepare Lysis Buffer 1. This should be pH 9 and comprised of the following reagents:

 
 
 

 

 
 
SECTION: Collection of larvae
4. Prepare 5 ml screwcap collection tubes containing one 4 or 4.5 mm hardened steel bead and 2ml of Lysis Buffer 1. All volumes for sample and homogenisation and digestion assume you are using a 5 ml screwcap tube. See notes below for further detail.
 

 

SECTION: Collection of larvae
2. 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 the pouring lip and lid removed then bake for at least 12 hours at 250 oC. 

  

 

SECTION: Collection of larvae
1. Source steel beads (ball bearings) for tissue grinding (Tungsten beads are not usually necessary for caterpillars). We use hardened carbon steel or stainless steel bearings from simplybearings.co.uk. This protocol requires one bead per sample tube.
SECTION: DNA extraction: purification
18.