TITLE: 18S rDNA amplification of Kudoa musculoliquefaciens in broadbill swordfish
AUTHORS: Jessica A Bolin

[DESCRIPTION]
Protocol for extracting and purifying genomic DNA from Kudoa musculoliquefaciens infecting broadbill swordfish Xiphias gladius, harvested off Eastern Australia. Used in Bolin et al. 2021 Parasitol. Res. (link) and Bolin et al. 2023 in review.  

[BEFORE_START]
Sanitise work area and equipment with 70% ethanol. 
Preheat heat block to 56˚C.
If using the DNA extraction kit for the first time, add the specified amount of 100% ethanol to the AW1 and AW2 buffers (will be on the protocol sheet and buffer container). Shake thoroughly after adding ethanol.
Prepare DNeasy Mini Spin Columns by placing them in associated 2mL collection tubes

[GUIDELINES]
Kudoid gDNA and PCR reagents must be kept on ice or at 4˚C throughout the protocol. 
Best results are obtained with fresh swordfish, or swordfish that has been snap-frozen and stored at -20˚C.
Avoid repeated freezing and thawing of samples. 
If using frozen tissue, equilibrate the sample to room temperature before commencing protocol. 
All centrifugation steps are carried out at room temperature (15–28˚C).


[STEPS]
SECTION: Extraction and purification of genomic DNA
1. Cut ~15mg of swordfish muscle tissue into small pieces, and place in a microtube. 
SECTION: Extraction and purification of genomic DNA
2. Add 180µl of ATL buffer to the sample. 
SECTION: Extraction and purification of genomic DNA
3. Add 20µl of proteinase K and vortex for 10 sec. 
SECTION: Extraction and purification of genomic DNA
4. Incubate at 56˚C in the heat block, until the sample is completely lysed (generally takes ~2 hours). Throughout lysis, vortex the sample for 10 sec every 30 minutes.
 

SECTION: Extraction and purification of genomic DNA
5. After ~2 hours of lysis, vortex the sample for 15 sec.
SECTION: Extraction and purification of genomic DNA
6. Add 200µl of AL buffer and 100% ethanol to the sample. 
SECTION: Extraction and purification of genomic DNA
7. Vortex for 10 sec.
SECTION: Extraction and purification of genomic DNA
8. Centrifuge 8000 rpm, 1 min 

SECTION: Extraction and purification of genomic DNA
9. Transfer the supernatant into the DNeasy Mini Spin Column + collection tube
SECTION: Extraction and purification of genomic DNA
10. Centrifuge 8000 rpm, 1 min 
SECTION: Extraction and purification of genomic DNA
11. Ensure that the supernatant has fallen through the spin column membrane. If not, this means the sample has not lysed properly and has clogged the spin column membrane. If this happens, either:
(i) Repeat Step 10
(ii) Start protocol from scratch and increase the incubation time. 
SECTION: Extraction and purification of genomic DNA
12. Discard flow-through and collection tube. Place spin column into a new 2mL collection tube
SECTION: Extraction and purification of genomic DNA
13. Add 500µL AW1 buffer
SECTION: Extraction and purification of genomic DNA
14. Centrifuge 8000 rpm, 1 min 
SECTION: Extraction and purification of genomic DNA
15. Discard flow-through and collection tube. Place spin column into a new 2mL collection tube.
SECTION: Extraction and purification of genomic DNA
16. Add 500µL AW2 buffer.
SECTION: Extraction and purification of genomic DNA
17. Centrifuge 14000 rpm, 3 min 
SECTION: Extraction and purification of genomic DNA
18. Ensure that the supernatant has fallen through the spin column membrane and there is no residual buffer/gDNA + the membrane is completely dry. This ensures that no residual buffer will be carried over during the following elution.
 

SECTION: Extraction and purification of genomic DNA
19. Discard flow-through and collection tube. Place spin column into a new 1.5mL microtube.
SECTION: Extraction and purification of genomic DNA
20. *Slowly and carefully* add 200µL AE buffer to the centre of the spin column. 
SECTION: Extraction and purification of genomic DNA
21. Incubate at room temperature for 1 min.
SECTION: Extraction and purification of genomic DNA
22. Centrifuge 8000 rpm, 1 min for final gDNA elution. 
SECTION: Extraction and purification of genomic DNA
23. Store purified DNA sample in 4˚C/-20˚C, or proceed to next section. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
24. Open Nanodrop 2000 software -> click nucleic acid/DNA -> wait for software to automatically calibrate
SECTION: Quantify DNA concentration, purity and evidence of degradation
25. Open the spectrophotometer probe and place 1µL of AE buffer onto the probe. Close the lid. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
26. Click the buffer icon. Spectrophotometer will commence measuring the buffer.
 

SECTION: Quantify DNA concentration, purity and evidence of degradation
27. Open the spectrophotometer probe, wipe away the AE buffer, and place 1µL of gDNA onto the hole. Close the lid. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
28. Click the measure icon. The concentration and purity readings will display on the screen.
 


SECTION: Quantify DNA concentration, purity and evidence of degradation
29. Close the Nanodrop software. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
30. Make up a 0.8X agarose gel. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
30.1. Add 0.8g of agarose power and 0.6X 100µL TBE buffer into a schott jar. Shake to mix.
SECTION: Quantify DNA concentration, purity and evidence of degradation
30.2. Microwave until agarose is completely dissolved.
SECTION: Quantify DNA concentration, purity and evidence of degradation
30.3. Allow jar to cool to room temperature, and place 1 drop of Ethidium Bromide into jar. Immediately close the lid and swirl gently. 
 

SECTION: Quantify DNA concentration, purity and evidence of degradation
30.4. Pour gel into a gel tray. Add a gel comb, and wait until the gel has completely cooled/solidified (~30-60 min).
SECTION: Quantify DNA concentration, purity and evidence of degradation
31. Mix 11µL of gDNA with 1µL of gel loading dye. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
32. Remove the gel comb from the tray. Insert gel tray into gel tank and submerge with 0.6X TBE. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
34. Run the gel at 100V for 1–2 hours. Stop the voltage when the bands have nearly run off the gel. 
SECTION: Quantify DNA concentration, purity and evidence of degradation
35. Visualise gel in the UV trans-illuminator. 
 

SECTION: Quantify DNA concentration, purity and evidence of degradation
36. Store gDNA sample in 4˚C/-20˚C, or proceed to next section. 
SECTION: PCR
37. Create a mastermix for a 25µL PCR reaction in a microtube, by combining the following reagents:
12.5µL of Taq ReadyMix
0.5µL of forward primer
0.5µL of reverse primer
1µL of gDNA
10.5µL of PCR pure water (or Ultrapure water)
 

SECTION: PCR
38. Add the mastermix into a PCR strip tube. Slowly pipette the mixture up and down to mix thoroughly. Repeat for as many samples as required. 
SECTION: PCR
39. Store samples at 4˚C or proceed to next step.
SECTION: PCR
40. Set thermocycler parameters to:
Initialization: 95˚C, 5 min
Denaturation: 95˚C, 30 sec
Annealing: 54.4˚C, 30 sec
Elongation/extension: 72˚C, 1 min
Repeat steps denaturation to elongation 35 times
Final elongation: 72˚C, 5 min
Hold: 10˚C (optional)
SECTION: PCR
41. Insert strip tubes into the thermocycler. Ensure tubes are spread evenly to balance the lid. Apply plastic/gel cover over the gel caps. 
SECTION: PCR
42. Close the lid and run the PCR. Will take ~2 hours.
SECTION: PCR
43. Store sample in 4˚C/-20˚C, or proceed to next section. 
SECTION: Electrophoresis
44. Make up 2X agarose gel
SECTION: Quantify DNA concentration, purity and evidence of degradation
33. Add 3µL of 1Kb DNA ladder to a lane, and the pre-mixed gDNA + dye to another lane.
SECTION: Electrophoresis
44.1. Mix 2g agarose powder and 100mL 0.6X TBE buffer in a schott jar. Shake to mix.
SECTION: Electrophoresis
44.2. Microwave until powder has completely dissolved. 
 

SECTION: Electrophoresis
44.3. Let gel cool to room temperature. Add 1 drop of Ethidium Bromide
SECTION: Electrophoresis
44.4. Pour gel into a gel tray and add gel comb. 
SECTION: Electrophoresis
44.5. When gel has completely set, remove gel comb. 
SECTION: Electrophoresis
45. Insert gel tray into gel tank. Submerge in 0.6X TBE buffer.
SECTION: Electrophoresis
46. Load 3µL of 1KB DNA ladder into a well. Load ~10µL of PCR reaction sample into other wells.
SECTION: Electrophoresis
47. Close tank lid and set voltage to 120V. Run gel for 30–40 min.
SECTION: Electrophoresis
48. Visualise gel on UV trans-illuminator.