TITLE: DNA extraction from fecal samples
AUTHORS: Anique Ahmad, Arya Gautam, Tsedenia Denekew, Aashish Jha

[DESCRIPTION]
DNA Extraction from fecal samples

[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.

[STEPS]
1. Transfer fecal sample to ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and add ZymoBIOMICS™ Lysis Solution:

0.21. 
1.1. Flash frozen fecal samples will be solid and an aliquot (15 mg) of feces is expected in a 2mL container (Table 1). Add 250 µL of ZymoBIOMICS™ Lysis Solution to the fecal sample and homogenize evenly using the homogenizing pestle while submerged in liquid nitrogen (or on dry ice). Once the sample is homogenized, add another 500 µL of ZymoBIOMICS™ Lysis Solution such that the final volume is 750 µL. Mix well by vortexing.
Spin the sample container for 10 s in a minicentrifuge.
Transfer the entire 750 µL of the sample mix into the ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and proceed to Step 2. 
1.2. Fecal samples collected using Zymo DNA/RNA Shield™ will exist as a solution (Table 1). Mix the sample by shaking well or vortexing. Add up to 250 µL of such sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). Adjust final volume to 1 mL with ZymoBIOMICS™ Lysis Solution. Cap the tube tightly and proceed to Step 2.
1.3. Fecal samples collected using other stabilization kits may exist as a solution (Table 1). Mix the sample by shaking well or vortexing. Add 250 µL of such sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). Add750 µL ZymoBIOMICS™ Lysis Solution to the tube. Cap tightly and proceed to Step 2.
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 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.