The D4309 ZymoBIOMICS DNA Kits are microbial DNA purification kits designed for purifying DNA from a variety of sample inputs that is immediately ready for microbiome or metagenome analyses. The ZymoBIOMICS lysis system eliminates bias associated with unequal lysis efficiencies of different organisms (e.g. Gram-negative/positive bacteria, fungus, protozoans, and algae), making it ideal for microbial community profiling. Uniform mechanical lysis of all microbes is achieved by bead beating with the innovative ultra-high density BashingBeads. This kit is equipped with our OneStep PCR Inhibitor removal technology, enabling PCR amplification from DNA derived from inhibitor-rich environmental samples. Purified DNA is ideal for all downstream applications including PCR, arrays, 16S rRNA gene sequencing, and shotgun sequencing. DNA Size is 15-20 kb.
|Applicable For||All sensitive downstream applications such as qPCR and Next-Generation Sequencing.|
|Elution Volume||≥ 20 µl DNA Elution Buffer or DNase free water|
|Equipment||Centrifuge w/ microplate carriers, 96-well plate/block disruptor or pulverizer|
|Processing Volume||Fecal ≤100 mg, Soil ≤ 100 mg, Cells ≤ 20 mg (Approximately equal to 2×108 bacterial, 2×107 yeast, or 2×106 mammalian cells)|
|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|
|Sample Source||Bacterial, fungal, protozoan, algae, viral, mitochondrial, and host DNA is efficiently isolated from feces, soil, fungal/bacterial cells, biofilms and water.|
|Sample Storage||Eluted DNA should be stored at ≤ -20°C.|
|Size Range||Typically 15-20 kb post-bead beating. For optimal DNA integrity, collect samples in DNA/RNA Shield.|
|Yield||Up to 5 µg total DNA can be eluted into 20 µl|
Q1: What is the purpose of the Zymo-Spin III-HRC Step?
Environmental samples often contain inhibitors such as polyphenolics, humic/fulvic acids, tannins, melanins, etc. that affect downstream applications such as PCR. The Zymo-Spin III-HRC is designed to remove these PCR inhibitors and does not bind DNA. The Zymo-Spin III-HRC can be purchased separately as the OneStep PCR Inhibitor Kit (D6030).
Q2: Why do I have low yield and/or low purity?
Check sample type and input amount. Low biomass samples such as swabs, some soil samples, water, air are expected to have low yield. High biomass samples such as feces may cause overloading of the spin column. Using less input may improve yields.
Ensure sample is fully homogenized. Depending on bead beater used, the settings may have to be optimized for each sample. The homogenization time may be extended to see if yields improve.
Apply heated elution buffer (60-70° C) and allow elution buffer to incubate on the column for several minutes prior to elution.
Ensure that the DNA Elution Buffer is added directly to the column matrix.
Low A260/230 values can be due to handling issues (e.g. transfer of column during wash steps). To minimize reagent wash buffer carryover, centrifuge the column at max speed for 1 minute prior to elution.
Q3: Debris on the column following binding step, what should I do to avoid this in the future?
Spin down lysate and Binding Buffer mixture at max speed for 5 minutes, this will eliminate any debris not caught by Spin III-F filter. Also avoid debris in lysis tube when transferring lysate, spin at max speed after bashing for 1-3 minutes.
Q4: Are there any tips in optimizing bead beating conditions?
We have validated our kits with both high-speed homogenizers and low-speed disruptors. We highly recommend users to optimize their bead beating conditions for their own instruments. We recommend using a 2 ml-tube adapter to ensure that the bead beating is efficient (do not use adapters made of foam). You can reference the Optimized Lysis Protocols table under documents for some recommendations.
Q5: What is the purpose of the Zymo-Spin III-F Filter?
This is to remove any debris from the lysate that could interfere with the DNA binding to the silica membrane of the column.