TITLE: DNA extraction from infected or not wheat leaf tissue
AUTHORS: Benjamin Schwessinger

[DESCRIPTION]
This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". 

You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments.

In total, each group will obtain five pots each:
 
 
This specific protocol is a step by step guide to sample representative leaf material for each treatment group and to extract DNA from these samples with the Qiagen DNeasy Plant Mini Kit. We use a kit in the course to speed up the process and make it reliable. Though it is important to understand the rough principles of DNA extraction as this process might influence the outcome of pathogen detection and identification in a biosecurity setting. We provide a short description of each step and its function in the protocol. In addition, please consult the handbook of the DNeasy Plant Mini Kit provider for further details as required.

The final goal is to achieve the following:
sample representative leaves with symptoms for each treatment group [TG1-TG4]  that contain enough infective agent for DNA based pathogen identification.
sample representative leaves without symptoms for TG5.
extract pure and high quality DNA that allows for PCR amplification of marker gene regions (bacteria and/or fungi) and direct DNA sequencing of total DNA.

This protocol is applicable for week 2. 

Protocols progress overview:

Week 2 Sampling of two representative leaves and subsections thereof.
Wee 2 tissue rupture with metal beads when tissues is frozen (performed by demonstrator).
Week 2 DNA extraction from ruptured leaf tissue.
 
The whole process follows the following major steps.

Sampling -> Tissue rupture -> Tissue lysis -> Clearance of material -> DNA precipitation -> DNA binding to silica matrix -> Washing of impurities -> DNA elution from column and collection. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

[BEFORE_START]
You must study the protocol carefully before you start. If anything is unclear post questions directly here on protocols.io.

Additional protocol specific notes:

Perform all centrifugation steps at room temperature (15–25°C).
If necessary, redissolve any precipitates in Buffer AP1 and Buffer AW1 concentrates (done by demonstrators).
Add ethanol to Buffer AW1 and Buffer AW2 concentrates  (done by demonstrators).
Preheat a water bath or heating block to 65°C (done by demonstrators).

[GUIDELINES]
You must have read, understood, and follow the health and safety instructions provided in the "Overview Mini-Research Project #1 BIOL3106/6106" provided on Wattle (ANU learning portal). 

You must have signed and returned one copy of the "Student Safety Declaration Form For Practical Class Work" before starting the any laboratory work. 

You must have read and understood the Hazard Sheets (Risk assessment) of all chemicals listed bellow in the "Safety Warnings" section. These Hazard Sheets are provided on Wattle as part of the  "Overview Mini-Research Project #1 BIOL3106/6106" document.

[STEPS]
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
1. Label each 2ml Eppendorf tube containing 2-3 steal beads with the treatment group name and your research group name. Label both the top and the side of each tube clearly.
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
2. Carefully, study the plants in each pot for potential disease symptoms. Select two leaves for each treatment group that show representative symptoms [TG#1-4] or not in the case of the negative control [TG#5].
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
2.1. Cut 1.5- 2 cm long leaf pieces from each TG from two independent leaves. Cut the middle part of the leaves. Combine both leaf pieces into the correctly labelled tube.
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
2.3. Once done with all five TGs, make sure all are frozen in liquid nitrogen with the demonstrator. The demonstrator will collect all tubes for one bench row before processing for tissue rupture.
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
2.2. Put the tube containing the leaf material on ice or freeze them immediately on liquid nitrogen handled by the demonstrator. 
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
3. The demonstrator will rupture your tissue using a TissueLyzer II https://www.qiagen.com/us/products/human-id-and-forensics/automation/tissuelyser-ii at 25 Hz for 1 min.
Now the leaf rupture part of the protocol is complete.
SECTION: Week 2: Sampling of representative leaf tissue and tissue rupture
2.4. Now the leaf tissues sampling part of the protocol is complete.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
4. Label all provided tubes and spin columns with your treatment group name and your research group name.
This implies to label the following set of tubes for each TG.
QIAshredder spin column (pinkish color) placed in a 2 ml collection tube. 
One additional 2m collection tube.
Two additional 1.5ml eppendorf tubes.
DNeasy Mini spin column (blueish color) placed in a 2 ml collection tube.

Now you will have five sets of clearly labeled tubes. This readies you to perform the DNA extraction for all five TG in parallel. 

SECTION: Week 2: DNA extraction from ruptured leaf tissue
5. You will receive your five tubes that now contain ruptured tissue on ice. It is import to resuspend the ruptured leaf tissue for all samples immediately in buffer to protect the DNA from DNAses without letting the tissue thaw in the tubes in the absence of buffer. The DNA will be protected in the AP1 buffer. Process all five samples at the same time.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.1. Add 400 μl Buffer AP1 to each of 2ml tubes containing your ruptured tissue. 
Steps 5.1 to 5.7 will lyse the cells of the ruptured tissue and bring the DNA into solution. The DNA is protected in the solution.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.2. Vortex and completely re-suspended the tissue in the AP1 buffer.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.4. Add 4 μl RNase A to each tube and vortex again. 
This enzyme will digest RNA that gets released during cell lysis. It performs best at  65°C.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.5. Incubate for 10 min at 65°C in the waterbath or heating block. 
Invert the tube 2–3 times during incubation.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.6. Collect content of the tube with a short "hand centrifuge" as demonstrated by the demonstrator.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.8. To each tube add 130 μl Buffer P3. Mix by inversion for 10 to 20 times immediately after adding the P3 buffer.
Steps 5.8 to 5.16 will clear the lysate so it is ready to precipitate the DNA without precipitating too many other contaminants. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.3. Collect content of the tube with a short "hand centrifuge" as demonstrated by the demonstrator. Hold the top of the tube between you fingers and pretend to 'throw' the tube to the ground but don't let go. This kind of rapid motion will collect most content at the bottom of the tube. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.7. Cool tubes on ice for 2 min. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.9. Incubate the five tubes on ice for 5 min. Slightly longer incubation will not affect the outcome negatively.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.10. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.11. Centrifuge the lysate for 5 min at maximum speed. 
This steep will clear the DNA in solution from larger undissolved particles.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.12. For each of your TG, pipett the 480 ul of the lysate into a QIAshredder spin column placed in a 2 ml collection tube. To not disturb the pellet when transferring the lysate. Remember! All tubes and spin columns must be labelled clearly.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.13. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.14. Centrifuge for 2 min at max speed.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.15. Keep the flow through in the lower collection tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.16. Discard the QIAshredder spin column.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.17. For each of your TG, transfer 400 ul of the flow-through into a new clearly labelled 1.5 ml tube without disturbing the pellet if present. 
The steps 5.17 and 5.18 will precipitate the DNA.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.18. For each TG, add 600 ul Buffer AW1, and mix by pipetting.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.19. Transfer 500 μl of the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Remember! All tubes and spin columns must be labelled clearly.
The steps 5.19 to 5.22 will bind the DNA to the silica membrane that is part of the DNeasy Mini spin column.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.20. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.21. Centrifuge for 1 min at max speed. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.22. Discard the flow-through by pouring it into the waste collection container.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.23. Place the DNeasy Mini spin column in the same 2ml collection tube.
The steps 5.23 to 5.45 wash the bound DNA from co-precipitated and bound impurities.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.24. For each TG, add the remaining sample of about 500 ul onto the correct DNeasy Mini spin column.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.25. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.26. Centrifuge for 1 min at max speed. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.27. Discard the flow-through by pouring it into the waste collection container.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.28. Place the spin column into a new clearly labeled 2 ml collection tube. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.29. Discard the old collection tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.30. For each TG, add 500 μl Buffer AW2 on top of the DNeasy Mini spin column. Do not touch the spin column with your pipette tip.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.31. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.32. Centrifuge for 1 min at max speed. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.33. Discard the flow-through by pouring it into the waste collection container.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.34. Tap the collection tube on a paper towel to remove the remaining liquid.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.35. Place the DNeasy Mini spin column in the same 2ml collection tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.36. For each TG, add 500 μl Buffer AW2 on top of the DNeasy Mini spin column. Do not touch the spin column with your pipette tip.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.37. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.38. Centrifuge for 2 min at max speed.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.39. Discard the flow-through by pouring it into the waste collection container.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.40. Tap the collection tube on a paper towel to remove the remaining liquid.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.41. Place the DNeasy Mini spin column in the same 2ml collection tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.42. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.43. Centrifuge for 1 min at max speed. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.44. Remove the DNeasy Mini spin column from the collection tube carefully so that the column does not come into contact with the flow-through.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.45. Transfer the spin column to a new clearly labelled 1.5 ml tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.46. Add 75 μl Buffer AE into the centre of the DNeasy Mini spin column for elution. Make sure to pipette the buffer directly onto the membrane in the centre of the tube.
The steps 5.46 to 5.56 elute the bound DNA from the silica membrane and bring it back into solution. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.47. Incubate for 5 min at room temperature (15–25°C). 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.48. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.49. Centrifuge for 1 min at max speed. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.50. Add another 75 μl Buffer AE into the centre of the DNeasy Mini spin column for elution. Make sure to pipette the buffer directly onto the membrane in the centre of the tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.51. Incubate for 5 min at room temperature (15–25°C). 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.52. Team up with other research groups to centrifuge your samples. Make sure all your tubes are clearly labelled with your research group name and treatment group name. Make sure the centrifuge is balanced before you start. Check with a demonstrator about centrifuge usage if you are unclear.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.53. Centrifuge for 1 min at max speed. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.54. Discard the DNeasy Mini spin column. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.55. Close and keep the collection tube.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
5.56. The DNA is now contained in your in your elute. 
SECTION: Week 2: DNA extraction from ruptured leaf tissue
6. You should have now five clearly labelled collection tubes with about 150ul DNA in elution buffer. Each tube contains the DNA of one TG.
SECTION: Week 2: DNA extraction from ruptured leaf tissue
6.1. Hand over your five clearly labelled tubes to the demonstrator for storage till next week.