The ZymoBIOMICS™ DNA Microprep Kit is designed for purifying DNA from a wide array of sample inputs (e.g. feces, soil, water, and biofilms), that is immediately ready for microbiome or metagenome analyses. The ZymoBIOMICS™ innovative lysis system eliminates bias associated with unequal lysis efficiencies of different organisms (e.g. Gram-negative/positive bacteria,fungus, protozoans, and algae)1 making it ideal for microbial community profiling. Unbiased mechanical lysis of tough microbes is achieved by bead beating with the innovative ZymoBIOMICS™ ultra-high density BashingBeads™ and validated using the ZymoBIOMICS™ Microbial Community Standard2, as shown in Figure 3. In addition, the ZymoBIOMICS™ DNA Micro kit is equipped with Zymo’s Proprietary OneStep™ PCR Inhibitor Removal technology enabling PCR from the most PCR prohibitive environmental samples rich in humic and fulvic acids, tannins, melanin, and other polyphenolic compounds. Coupling state-of-the-art lysis technology with Zymo-Spin™ purification technology results in superior yields of ultra-pure DNA ideal for all downstream applications including PCR, arrays, 16S rRNA gene sequencing, and shotgun sequencing.
|DNA Recovery||Up to 5 μg total DNA can be eluted into 10 μl (6 μl minimum) ZymoBIOMICS™ DNase/RNase Free Water.|
|Equipment||Microcentrifuge, vortex/Disruptor Genie®, high speed cell disrupter (recommended).|
|Sample Sources||Bacterial, fungal, viral and other microbial DNA can be isolated from 5-20 mg (wet weight) of fungal/bacterial cells|
|DNA Purity||High quality, inhibitor-free DNA is eluted with ZymoBIOMICS™ DNase/RNase Free Water and is suitable for all downstream applications including PCR and Next-Generation sequencing|
|Bead Beating System||The ZymoBIOMICS™ innovative lysis system enables complete homogenization/disruption of the microbial cells walls and accurate microbial DNA analysis, free of bias. To ensure unbiased lysis it is recommend that the ZymoBIOMICS™ Microbial Community Standard is used for calibration of each bead beating device|
|DNA Integrity||Generally, post bead beating, genomic DNA has an average size of 15-20 kb depending on the initial quality of the sample making it amenable to Next-Gen sequencing platforms requiring high molecular weight DNA. For optimal DNA integrity, collect samples in DNA/RNA Shield™|
|Bioburden||A single preparation is guaranteed to contain less than 3 bacterial genomic copies per 1 μl of eluate as determined by quantitative amplification of the 16S rRNA gene when eluted using 100 μl water.|
|Figure 1. The ZymoBIOMICS™ -96 Magbead DNA Kit produces linear recovery of DNA for sensitive applications, detecting pathogenic organisms such as E. coli and H. pylori in assays with up to a 1000x dilution factor. A dilution series of stool spiked with 1x106 E. Coli cells and H. Pylori infected stool was extracted using the ZymoBIOMICS™ - 96 Magbead DNA Kit, showing effective purification and qPCR amplification, even at 1000:1 dilution. Healthy stool sample controls did not amplify pathogenic organisms compared to the spiked stools.||Figure 2. The ZymoBIOMICS™ DNA Mini Kit provides inhibitor-free DNA even when challenged with extremely inhibitor rich samples. Real-time PCR was used to evaluate eluates recovered using the ZymoBIOMICS™ DNA Mini Kit, or Suppliers M, P, and Q. Reaction volumes consisted of either 10% or 35% of the eluate from each kit to detect the presence of PCR inhibitors. Each reaction contained 25 ng of Brettanomyces DNA. Delayed and/or no amplification indicates PCR inhibition from inefficient inhibitor removal.|
|Figure 3. The ZymoBIOMICS™ DNA Mini Kit provides superior yields when compared to Suppliers M, P, and Q. 80 mg of feces was processed using each kit according to the manufactures’ recommended protocol. DNA was eluted using 100 µl. 6 µl of each sample was analyzed in a 1.0% (w/v) agarose/ethidium bromide gel. Samples were processed in triplicate. L is a 1Kb ladder.|
|A) Bias Free Microbial DNA Extraction Using ZymoBIOMICS™ DNA Mini Kit Validated with the ZymoBIOMICS™ Microbial Community Standard||B) Bias Free Microbial DNA Extraction Using ZymoBIOMICS™ DNA Mini Kit From Human Stool|
|A) The ZymoBIOMICS™ DNA Mini Kit provides unbiased representation of the organisms extracted from the ZymoBIOMICS™ Microbial Community Standard. DNA was extracted from ZymoBIOMICS™ Microbial Community Standard using four different DNA extraction methods (ZymoBIOMICS™ DNA Mini Kit, Human Microbiome Project Protocol, Supplier M, and Supplier Q) and analyzed using 16S rRNA gene sequencing. 16S rRNA genes were amplified with primers targeting v3-4 region and the amplicons were sequenced on Illumina® MiSeq™ (2x250bp). Overlapping paired-end reads were assembled into complete amplicon sequences. The composition profile was determined based on sequence counts after mapping amplicon sequences to the known 16S rRNA genes of the eight different bacterial species.||B) The ZymoBIOMICS™ DNA Mini Kit reliably isolates DNA from even the toughest to lyse gram positive organisms, enabling unbiased analyses of microbial community compositions. There is a significant increase in yield and Gram-positive abundance when DNA was isolated using the ZymoBIOMICS™ DNA Mini Kit. Correlated with the results in Figure 3A it can be concluded that unbiased DNA isolation was achieved. DNA was extracted from 200 µl of human feces suspended in PBS (10 % m/v) using four different DNA extraction methods (ZymoBIOMICS™ DNA Mini Kit, Human Microbiome Project Protocol, Supplier M, and Supplier Q) and analyzed using 16S rRNA gene sequencing. 16S rRNA genes were amplified with primers targeting v3-4 region and the amplicons were sequenced on Illumina® MiSeq™ (2x250bp). Overlapping paired-end reads were assembled into complete amplicon sequences. Amplicon sequences were profiled with Qiime using Greengenes 16S rRNA gene database (gg_13_8).