DNA extraction for fermented plant based foods
Anique Ahmad, Tsedenia Denekew, Arya Gautam, Ahmed Shibl, Aashish Jha
Abstract
DNA Extraction for fermented plant based foods
Before start
Label the following: ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)
ZymoSpin<sup>TM</sup>III-F Filter in Collection Tube
Collection Tube
ZymoSpin<sup>TM</sup>II-CR Column in Collection Tube
ZymoSpin<sup>TM</sup>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.
Steps
Take 250mg 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) . Add750µL ZymoBIOMICS™ Lysis Solution to the tube. Cap tightly.
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 0h 5m 0s . Rest for 0h 5m 0s .
Centrifuge the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) in a microcentrifuge at ≥ 10000x g.
Transfer up to 600µL supernatant to the Zymo-Spin™ III-F Filter in the labelled Collection Tube and centrifuge at 8.000x g. Discard the Zymo-Spin™ III-F Filter.
Add 600µL of ZymoBIOMICS™ DNA Binding Buffer to the filtrate in the labelled Collection Tube from Step 4. Mix well.
Repeat Step 5 so that the final volume of ZymoBIOMICS™ DNA Binding Buffer added is 1200µL .
Transfer 800µL of the mixture from Step 5 to a Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10.000x g.
Discard the flow through from the Collection Tube and repeat Step 6.
Add 400µL ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™ IICR Column in a new Collection Tube and centrifuge at 10.000x g. Discard the flow-through.
Add 700µL ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10.000x g. Discard the flow-through.
Repeat with 200µL ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10.000x g.
Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml microcentrifuge tube and add 100µL (50 µl minimum) ZymoBIOMICS™ DNase/RNase Free Water directly to the column matrix and incubate for 0h 5m 0s. Centrifuge at 10.000x g to elute the DNA
Place a Zymo-Spin™ III-HRC Filter in a new Collection Tube and add 600µL ZymoBIOMICS™ HRC Prep Solution. Centrifuge at 8.000x g.
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 16.000x g.
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)
