About Quick-DNA™ Fecal/Soil Microbe Midiprep Kit
The ZR Fecal DNA MidiPrep™ (D6110) has been updated to Quick-DNA™ Fecal/Soil Microbe Midiprep Kit; all components and protocols are the same, Catalog numbers and names of components may have been modified but original buffer recipe remains the same.
The Quick-DNA™ Fecal/Soil Midiprep Kit 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||Typically, up to ~125 µg total DNA is eluted into ≥ 150 µl DNA Elution Buffer per sample.|
|Processing Time||25 min|
|Equipment||Centrifuge, Vacuum Source and Manifold, Microcentrifuge, Cell Disrupter/Pulverizer w/ 50 ml Tube Adapter|
|DNA Size Limits||Capable of recovering genomic DNA sized fragments from 100 bp to ≥40 kb. Typical fragment sizes range from 25 to 35 kb. If present, parasitic and viral DNA will also be recovered.|
|Sample Sources||Bacterial, protist, as well as host DNA can be effectively isolated from a ≤375 mg sample of mammalian feces.|
|DNA Purity||High quality, humic/fulvic-free DNA is eluted with DNA Elution Buffer making it perfect for PCR. A[260nm/280nm] >1.8, A[260nm/230nm] >2.0|
Comparison of DNA yields from rat feces using the Quick-DNA™ Fecal/Soil Microbe Midiprep Kit 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.
PCR of DNAs from rat and human fecal samples isolated with the Quick-DNA™ Fecal/Soil Microbe Midiprep Kit. Panels A and B show the results of PCR with DNA isolated from rat and human fecal samples, respectively, using primers specific for prokaryotic 16S rRNA. Panel C shows the results of PCR of DNA isolated from human feces with and without the addition of E. coli containing pUC19 plasmid DNA (indicated at the top of the image) using primers specific for the pUC19 sequence. In each case, amplicons were analyzed in a 1.5% (w/v) agarose / ethidium bromide gel using a UV imager. Numbers above each lane of the gel images are the volumetric equivalent (in µl) of eluted DNA (100 µl) used for PCR. Arrows mark the relative migration of amplicons in the gels, and M is a 100 bp DNA ladder (NEB).
The Quick-DNA™ Fecal/Soil Microbe Midiprep Kit can be used to isolate high quality DNA from a variety of soil types which yields robust products following PCR. Panel A: Physical characteristics of sampled soils (1-5) (Ref. 1). Panel B: Microbial DNA was isolated from soil samples (1-5) using the Quick-DNA™ Fecal/Soil Microbe Midiprep Kit. Approximately 10% of the eluted DNA was then separated in a 0.8% (w/v) agarose/ethidium bromide gel. Panels C and D show the results of PCR of microbial DNA isolated from the samples with primers specific for prokaryotic 16S rRNA (C) or eukaryotic rRNA (D). In the figures, the 1 kb size marker (NEB) is as indicated and the arrows show the prokaryotic 16S rRNA and eukaryotic rRNA PCR products.
DNA isolated from Saccharomyces cerevisiae (strain TMY18) using the Quick-DNA™ Fecal/Soil Microbe Midiprep Kit is high-quality and structurally intact. Equivalent amounts of yeast were processed using the Quick-DNA™ Fecal/Soil Microbe Midiprep Kit or the kits from suppliers M and E. Equal volumes of eluted DNA were then analyzed in a 0.8% (w/v) agarose/ethidium bromide gel. The size marker is a 1 kb ladder (NEB).
Add up to 375 mg of fecal sample to the bead/filter chamber of a ZR BashingBead™ Lysis/Filtration Tube. Add 6 ml Fecal DNA Binding Buffer to the sample, cap tube1, and process.
(To prevent the Lysis Solution from leaking into the bottom of the 50 ml tube, place the ZR BashingBead™ Lysis/Filtration Tube on its side prior to processing).
Secure in a bead beater fitted with a 50 ml tube holder assembly (see page 6) to process samples. Optimization of processing time/speed will be necessary for complete sample lysis.
Processing times may be as little as 40 seconds when using high-speed cell disrupters (e.g., FastPrepâ-24, Geno/Grinderâ, page 6). See manufacturer’s literature for operating information.
Centrifuge the ZR BashingBead™ Lysis/Filtration Tube in a centrifuge at ≥3,000 x g (5,000 x g max.) for 5 minutes.
Filter the entire mixture from Step 3 using a Zymo-Spin™ V-E Column/Zymo-Midi Filter™ assembly mounted on a vacuum manifold2,3 (see diagram on page 2) with a vacuum source set at ≥600 mm Hg.
Disconnect the Zymo-Spin™ V-E Column/Zymo-Midi Filter™ assembly and transfer the Zymo-Spin™ V-E Column to a Collection Tube. Spin the column at 10,000 x g for 1 minute in a microcentrifuge4, then add 300 µl DNA Pre-Wash Buffer to the column and spin at 10,000 x g for 1 minute. Discard the flow through.
Add 400 µl Fecal DNA Wash Buffer to the column and centrifuge at 10,000 x g for 1 minute. Discard flow through and repeat wash step.
Transfer the Zymo-Spin™ V-E Column to a 1.5 ml microcentrifuge tube and add 150 µl DNA Elution Buffer directly to the column matrix5 and allow column to stand for 1 minute at room temperature. Centrifuge at 10,000 x g for 1 minute to elute the DNA6.
Transfer the eluted DNA from Step 7 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 suitable for PCR, other downstream applications, or storage.