DNA Extraction with ZymoBIOMICS MiniPrep Kit

Add Sampleto a ZR BashingBead^TM Lysis Tubes (0.1 & 0.5 mm). Add750µL ZymoBIOMICS^TM Lysis Solution to the tube and cap tightly. Note: For samples stored and lysed in Lysis Tubes, do not add ZymoBIOMICS^TM Lysis Solution and proceed to Step 2.

A	B
Sample Type	Maximum Input
Feces	200 mg
Soil	250 mg
Liquid Samples and Swab Collections	250 ul
Cells (isotonic buffer, el.g., PBS)	50-100 mg (wet weight) (109 bacterial and 108 yeast cells)
Samples in DNA/RNA Shield	<1 ml

Table from ZymoBIOMICS Protocol D4300

Obtain DNA/RNA Shield Lysis Tubes with samples/swab heads from instructors. Secure in a bead beater fitted with a 2 ml tube holder assembly and process using optimized beat-beating conditions (speed and time) for different devices. We will use a Vortex Genie with 2ml Bashing Bead tubes for 0h 40m 0s.

Centrifuge the ZR BashingBead^TM Lysis Tubes (0.1 & 0.5 mm) in a microcentrifuge at ≥ 10000rpm,20°C.

Transfer up to 400µL supernatant to the Zymo-Spin^TM III-F Filter in a Collection Tube and centrifuge at 8.000x g. Discard the Zymo-Spin^TM III-F Filter.

Add 1200µL of ZymoBIOMICS^TM DNA Binding Buffer to the filtrate in the Collection Tube from Step 4. Mix well.

Transfer 800µL of the mixture from Step 5 to a Zymo-Spin^TM 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^TM DNA Wash Buffer 1 to the ZymoSpin^TM IICR Column in a new Collection Tube and centrifuge at 10.000x g. Discard the flow-through.

Add 700µL ZymoBIOMICS^TM DNA Wash Buffer 2 to the ZymoSpin^TM IICR Column in a Collection Tube and centrifuge at 10.000x g. Discard the flow-through.

Add 200µL ZymoBIOMICS^TM DNA Wash Buffer 2 to the ZymoSpin^TM IICR Column in a Collection Tube and centrifuge at 10.000x g.

Transfer the Zymo-Spin^TM IICR Column to a clean 1.5 ml microcentrifuge tube and add 100 µl (50 µl minimum) ZymoBIOMICS^TM DNase/RNase Free Water directly to the column matrix and incubate for 0h 1m 0s. Centrifuge at 10.000x g to elute the DNA5, 6.

Place a Zymo-Spin^TM III-HRC Filter in a new Collection Tube and add 600µL ZymoBIOMICS^TM HRC Prep Solution. Centrifuge at 8.000x g.

Transfer the eluted DNA (Step 11) to a prepared Zymo-Spin^TM 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.