DNA extraction from fecal samples
Anique Ahmad, Tsedenia Denekew, Arya Gautam, Aashish Jha
Abstract
DNA Extraction from fecal samples
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
Transfer fecal sample to ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and add ZymoBIOMICS™ Lysis Solution:
Flash frozen fecal samples will be solid and an aliquot ( ) of feces 15mg) of feces is expected in a 2mL container (Table 1). Add 250µL 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 500µL of ZymoBIOMICS™ Lysis Solution such that the final volume is 750µL. Mix well by vortexing.* Spin the sample container for 0h 0m 10s in a minicentrifuge.
- Transfer the entire
750µLof the sample mix into the ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and proceed to Step 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 250µL of such sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) . Adjust final volume to 1mL with ZymoBIOMICS™ Lysis Solution. Cap the tube tightly and proceed to Step 2.
Fecal samples collected using other stabilization kits may exist as a solution (Table 1). Mix the sample by shaking well or vortexing. Add 250µL of such sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) . Add750µL ZymoBIOMICS™ Lysis Solution to the tube. Cap tightly and proceed to Step 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 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 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)
