TITLE: DNA extraction for fermented plant based foods
AUTHORS: Anique Ahmad, Arya Gautam, Tsedenia Denekew, Ahmed Shibl, Aashish Jha

[DESCRIPTION]
DNA Extraction for fermented plant based foods

[BEFORE_START]
Label the following:
    ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)
    ZymoSpinTM III-F Filter in Collection Tube
    Collection Tube
    ZymoSpinTM II-CR Column in Collection Tube
    ZymoSpinTM III-HRC Filter in Collection Tube
    2 Sets of 1.5 ml microcentrifuge tubes (not provided with kit)
Spin the labelled ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm) for 10 seconds in a mini-centrifuge to ensure that the beads have settled at the bottom
Include 1 control per batch and assign its position randomly.

[GUIDELINES]
Disinfect mortar and pestle with 70% ethanol in between samples
If the goal is metagenomic sequencing, we recommend bypassing the mortar and pestle step. Simply suspend the samples in 1 ml of ZymoBIOMICS™ Lysis Solution, shake vigorously or vortex, and transfer 750 uL of the supernatant to  a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and proceed to Step 2.

[STEPS]
2. Secure in a Omni Bead Ruptor Elite bead beater fitted with a 2 ml tube holder assembly and process at max speed (30 m/s) for 5 min . Rest for 5 min .
3. Centrifuge the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) in a microcentrifuge at ≥ 10000 x g, 1 min.
4. Transfer up to 600 µL supernatant to the Zymo-Spin™ III-F Filter in the labelled Collection Tube and centrifuge at 8.000 x g, 1 min. Discard the Zymo-Spin™ III-F Filter.
5. Add 600 µL of ZymoBIOMICS™ DNA Binding Buffer to the filtrate in the labelled Collection Tube from Step 4. Mix well.
5.1. Repeat Step 5 so that the final volume of ZymoBIOMICS™ DNA Binding Buffer added is 1200 µL .
6. Transfer 800 µL of the mixture from Step 5 to a Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10.000 x g, 1 min.

6.1. Discard the flow through from the Collection Tube and repeat Step 6.
7. Add 400 µL ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™ IICR Column in a new Collection Tube and centrifuge at 10.000 x g, 1 min. Discard the flow-through.

8. Add 700 µL ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10.000 x g, 1 min. Discard the flow-through.

9. Repeat with 200 µL ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10.000 x g, 1 min.

10. Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml microcentrifuge tube and add 100 µL (50 µl minimum) ZymoBIOMICS™ DNase/RNase Free Water directly to the column matrix and incubate for 5 min. Centrifuge at 10.000 x g, 1 min to elute the DNA

13. The filtered DNA is now suitable for PCR and other downstream applications. Eluted DNA should be frozen (–30 to –15°C or –90 to –65°C)
11. Place a Zymo-Spin™ III-HRC Filter in a new Collection Tube and add 600 µL ZymoBIOMICS™ HRC Prep Solution. Centrifuge at 8.000 x g, 3 min.

12. Transfer the eluted DNA (Step 10) to a prepared Zymo-Spin™ III-HRC Filter in a clean 1.5 ml microcentrifuge tube and centrifuge at exactly 16.000 x g, 3 min.
1. Take 250 mg of food sample while on dry ice and transfer to mortar. Add liquid nitrogen until food sample is completely immersed. Using pestle vigorously homogenize sample until a powder forms. Transfer all of the sample to  a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). Add750 µL ZymoBIOMICS™ Lysis Solution to the tube. Cap tightly.