The ZymoBIOMICS DNA/RNA Miniprep Kit is designed for purifying DNA and RNA from a wide array of sample inputs (e.g. feces, soil, plant, water, and biofilms) that is 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). The provided DNA/RNA Shield preserves nucleic acids at ambient temperatures, providing an unbiased molecular snapshot of the sample. The procedure uses Zymo-Spin Column technology that results in high-quality DNA and total RNA (including small RNAs 17-200 nt) that is free of PCR inhibitors (e.g. polyphenols, humic acids, and fulvic acids). Ready for RT-PCR, arrays, sequencing, etc.
|Equipment||Microcentrifuge, vortex, cell disrupter (recommended)|
|Purity||DNA and RNA is ready for Next-Gen sequencing, RTPCR, microarray, hybridization, etc. A260/A280, A260/A230: >1.8.|
|Sample Source||Bacterial, fungal, protozoan, algae, viral, mitochondrial, and host RNA is efficiently isolated from ≤ 200 mg of mammalian feces, ≤ 250 mg soil, ≤ 200 mg plant/seed, 50-100 mg (wet weight) fungal bacterial cells, biofilms, and water.|
|Size Range||DNA and Total RNA ≥17 nt|
|Yield||100 µg DNA/RNA (binding capacity), ≥50 µl (elution volume)|
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/RNA. 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: I noticed a degraded RNA or intense band at <200 nt size, what is causes this?
The chemistry extracts total RNA (including small RNAs and miRNAs) so an intense band at <200 nt size is within reason. For actual degraded RNA, make sure to check the sample collection procedures and storage conditions to ensure the initial sample is high quality. Degraded RNA can also occur if the bead-beating sample tube is overheated during homogenization. Reducing the time and intensity may improve performance.
Q4: What do I do if I notice precipitation during ethanol addition?
Sample overloading can cause nucleic acids to precipitate and crash out of solution, reducing yields. Use less sample input to avoid precipitation.
Q5: What do I do if there is foaming of sample during homogenization?
This is a normal occurrence as DNA/RNA Shield contains detergents. To reduce foam, centrifuge at ≥ 12,000 x g in 1 minute intervals before transferring the supernatant.
Q6: 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.