About ZR Fecal DNA MiniPrep™
The ZR Fecal DNA MiniPrep™ is designed for the simple and rapid isolation of inhibitor-free, PCR-quality host cell and microbial DNA from a variety of sample sources including humans, birds, rats, mice, cattle, etc. The procedure is easy and can be completed in minutes: fecal samples are rapidly and efficiently lysed by bead beating with our state of the art, ultra-high density BashingBeads™. Fast-Spin column technology is then used to isolate the DNA which is subsequently filtered to remove humic acids/polyphenols that can inhibit PCR. Eluted DNA is ideal for downstream molecular-based applications including PCR, arrays, genotyping, methylation detection, etc.
|DNA Recovery||Up to 25 μg total DNA is eluted into ≥ 25 μl DNA Elution Buffer per sample.|
|Processing Time||15 min|
|Equipment||Microcentrifuge, vortex, cell disruptor/pulverizer (optional)|
|DNA Size Limits||Capable of recovering genomic DNA up to and above 40 kb. In most instances, mitochondrial DNA and viral DNA (if present) will also be recovered.|
|Sample Sources||Bacterial, protist, as well as host DNA can be effectively isolated from a ≤150 mg sample of mammalian feces.|
|DNA Purity||High quality, inhibitor-free DNA is eluted with DNA Elution Buffer suitable for the amplification of bacterial, protist, and/or mammalian templates. A260/A280 >1.8|
Comparison of DNA yields from rat feces using the ZR Fecal DNA MiniPrep™ and kits from suppliers A and B. Equivalent amounts of feces were processed using each kit and then equal volumes of eluted DNA were analyzed in a 0.8% (w/v) agarose/ethidium bromide gel. Samples were processed in triplicate.
Add up to 150 mg of fecal sample to a ZR BashingBead™ Lysis Tube. Add 750 µl Lysis Solution to the tube.
Secure in a bead beater fitted with a 2 ml tube holder assembly (e.g., Disruptor Genie™) and process at maximum speed for 5 minutes.
Processing times may be as little as 40 seconds when using high-speed cell disrupters (e.g., the portable Xpedition™ Sample Processor, page 6, FastPrepâ-24, or similar). See manufacturer’s literature for operating information.
Centrifuge the ZR BashingBead™ Lysis Tube in a microcentrifuge at ≥10,000 x g for 1 minute.
Transfer up to 400 µl supernatant to a Zymo-Spin™ IV Spin Filter (orange top) in a Collection Tube and centrifuge at 7,000 rpm (~7,000 x g) for 1 minute.
Add 1,200 µl of Fecal DNA Binding Buffer to the filtrate in the Collection Tube from Step 4.
Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IIC Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
Discard the flow through from the Collection Tube and repeat Step 6.
Add 200 µl DNA Pre-Wash Buffer to the Zymo-Spin™ IIC Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute.
Add 500 µl Fecal DNA Wash Buffer to the Zymo-Spin™ IIC Column and centrifuge at 10,000 x g for 1 minute.
Transfer the Zymo-Spin™ IIC Column to a clean 1.5 ml microcentrifuge tube and add 100 µl (25 µl minimum) DNA Elution Buffer directly to the column matrix. Centrifuge at 10,000 x g for 30 seconds to elute the DNA.
Transfer the eluted DNA from Step 10 to a prepared Zymo-Spin™ IV-HRC Spin Filter (green top) (see above) in a clean 1.5 ml microcentrifuge tube and centrifuge at exactly 8,000 x g for 1 minute. The filtered DNA is now suitable for PCR and other downstream applications.
High-quality DNA was purified from Blastocystis positive fecal samples using the ZR Fecal DNA MiniPrep™ for the purpose of comparing detective sensitivity between PCR methods and other commercial products. DNA was determined to be inhibitor free and highly useable for PCR analysis with STS primers and a partial SSU rRNA gene, outperforming all other kits tested.
The ZR Fecal DNA MiniPrep™ was identified as an optimal solution for obtaining total bacterial DNA from swine feces, outperforming all other kits tested in terms of DNA yield, DNA purity, DNA degradation, time of extraction process, and cost. Samples were processed successfully from both RNAlater® and liquid nitrogen storage.
Pakpour et. al. (2012) A multi-criteria decision-making approach for comparing sample preservation and DNA extraction methods from swine feces. American Journal of Molecular Biology.
DNA was purified using the ZR Fecal DNA MiniPrep™ and used with PCR for detecting Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples. Detection was 94% accurate by IS900 PCR, the highest of any other kit tested, demonstrating correct methodology for the most accurate detection.
Tritrichomonas foetus was detected by PCR after DNA extraction using the ZR Fecal DNA MiniPrep™, demonstrating the greatest analytical sensitivity and reproducibility of all other kits tested. Detection was 100% successful, identifying ≥10 foetus organisms per 100 mg of sample.