TITLE: Endophytic fungal DNA extraction from seeds using Qiagen DNeasy Plant Mini
AUTHORS: Eve Kenny

[DESCRIPTION]
This protocol was modified from the QIAGEN Plant Mini Kit Handbook to extract fungal DNA from within the seed coat of small (<1mm diameter) seeds. It can be followed by amplification with fungal specific primers (ITS-1F KY02 and ITS-4 KY01) to optimize concentration of fungal DNA. Together, this maximizes the ratio of fungal DNA to plant DNA for metagenomic analysis of the fungal community.

[BEFORE_START]
This protocol takes approximately 2 hours to process 8 seeds. Additional time per sample depends mostly on the capacity of the centrifuge and beadbeater. For example, as my beadbeater could only process 8 tubes at a time, anymore than 8 seeds doubled my time for "Surface sterilization and lysis", which was typically the longest section.

1. If Buffer AP1 and Buffer AW1 have formed precipitate in storage, redissolve at 65°C. Do not heat Buffer AW1 once ethanol has been added.
2. Add ethanol (>96%) to Buffer AW1 and AW2 as indicated on buffer bottle.
3. Preheat heat block to 65°C
4. Prepare ice bath

[GUIDELINES]
Refer to Qiagen DNeasy Plant Mini handbook for troubleshooting. I have written what worked best for my needs, but Qiagen provides detailed information about how to modify the procedure for different purposes.

The handbook recommends that less than 20 mg dry weight be used with the kit, but I have found that with fungal extraction from seeds, I have never exceeded this limit.

[STEPS]
SECTION: Surface sterilization and lysis
1. Rinse seeds in microcentrifuge tube with ddH20 and finger vortex for 1 minute. Replace water and repeat two more times. Move seeds to screw cap tubes and add a large spatula scoop of mini beads (~100 μg).
SECTION: Surface sterilization and lysis
2. Add 400 μg  Buffer AP1 to each tube and use micro-pestle to grind seed coat until seed interior is exposed (1 minute).
SECTION: Surface sterilization and lysis
3. Add tubes to the beadbeater and run for 1 minute. Follow with 1 minute in the centrifuge at 12,000 rpm. Repeat three times for a total of four rounds of beadbeating and spinning.

Note: This is the top speed of the Sigma 112 Mini Centrifuge. I have not tested if a different speed yields different results.
SECTION: Surface sterilization and lysis
4. Incubate the mixture for 10 minutes at 65°C. Mix three times during incubation by inverting tube.
SECTION: Surface sterilization and lysis
5. Repeat Step 3. Incubate for 10 minutes, or until foam has been brought down. 
SECTION: Surface sterilization and lysis
6. Spin the tubes briefly. Remove lysate and place in a new microcentrifuge tube.
SECTION: Lysate purification
7. Add 130 μL Buffer P3 to the lysate. Mix and incubate for 5 minutes on ice
SECTION: Lysate purification
8. Centrifuge the lysate for 5 minutes at 14,000 rpm.
SECTION: Lysate purification
9. Pipet the lysate into the QIAsheredder Mini spin column (lilac) placed in the supplied 2 mL collection tube. Discard pellet and old microcentrifuge tube. Centrifuge spin column in collection tube for 2 minutes at 14,000 rpm. 
SECTION: Lysate purification
10. Carefully pipet flow-through into a new microcentrifuge tube without disturbing the cell-debris pellet. Record amount of flow-through transferred (typically about 350 μL).
SECTION: Lysate wash
11. Add 1.5 x lysate volume of Buffer AW1 to lysate. Mix IMMEDIATELY by pipetting.

Example: 350 μL lysate recovered = 525 μL Buffer AW1
SECTION: Lysate wash
12. Pipet 650 μL mixture (including precipitate) into the DNeasy Mini spin column (white) placed in the supplied 2 mL collection tube. Centrifuge for 1 minute at 8000 rpm. 
SECTION: Lysate wash
13. Discard flowthrough and add 650 μL remaining mixture to the same column and collection tube. Repeat centrifugation for 1 minute at 8000 rpm. Discard flowthrough and repeat until all lysate is used up. Discard collection tube.
SECTION: Lysate wash
14. Place DNeasy Mini spin column into a new supplied 2 mL collection tube. Add 500 μL Buffer AW2 and centrifuge for 1 minute at 8000 rpm.
SECTION: Lysate wash
15. Discard flowthrough and add 500 μL Buffer AW2 to same column and collection tube. Centrifuge for 2 minutes at 14,000 rpm.

Note: This step dries the membrane.
SECTION: Elution
16. Transfer the DNeasy Mini spin column to a new microcentrifuge tube. Pipet 50 μL Buffer AE directly onto spin column membrane. Incubate for 5 minutes at room temperature and centrifuge for 1 minute at 8000 rpm.

Note: you can elute with 100 μL Buffer AE for a greater overall yield of DNA, yet this dilutes the final DNA concentration for each elution. Refer to DNeasy Plant Mini Handbook for more information.  
SECTION: Elution
17. Repeat step 16 in a new microcentrifuge tube. 

Note: You can elute into the same tube used in step 16, but I found that this diluted my DNA too much.