About Quick-DNA™ Fungal/Bacterial Miniprep Kit
Note: The ZR Fungal/Bacterial DNA MiniPrep™ (D6005) has been updated to Quick-DNA™ Fungal/Bacterial Miniprep 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™ Fungal/Bacterial Miniprep Kit is designed for the simple and rapid isolation of DNA from tough-to-lyse fungi, including A. fumigatus, C. albicans, N. crassa, S. cerevisiae, S. pombe, as well as Gram (+/-) bacteria, algae, and protozoa. The procedure is easy and can be completed in minutes: fungal and/or bacterial samples are rapidly and efficiently lysed with our state of the art, ultra-high density BashingBeads™. Fast-Spin column technology is then used to isolate the DNA that is ideal for downstream molecular-based applications including PCR, array, etc.
|DNA Recovery||Typically, up to 25 µg total DNA is eluted into ≥ 25 µl DNA Elution Buffer per sample.|
|Processing Time||10 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||50-100 mg (wet weight) fungi or bacteria or up to 200 mg tissue. This equates to approximately 109 bacterial cells, 108 yeast cells and 107 mammalian cells. Spores, pollen, nematodes, as well as other microorganisms can also be sampled.|
|DNA Purity||High quality DNA is eluted with DNA Elution Buffer making it perfect for PCR. A260/A280 >1.8|
Fungal and bacterial DNA purification. DNA isolated from Saccharomyces cerevisiae (spores) and E. Coli using the Quick-DNA™ Fungal/Bacterial Miniprep Kit is high quality and structurally intact. Equivalent amounts of yeast and bacteria were processed using the Quick-DNA™ Fungal/Bacterial Miniprep Kit or the kit from supplier A. 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 (Zymo Research).
Add 50-100 mg (wet weight) fungal or bacterial cells that have been resuspended in up to 200 µl of water or isotonic buffer (e.g., PBS) or up to 200 mg of tissue 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 5, FastPrep-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.
Snap off the base of the Zymo-Spin IV™ Spin Filter prior to use.
Add 1,200 µl of Fungal/Bacterial 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 Fungal/Bacterial 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.
Ultra-pure DNA is now ready for use in your experiments.
The ZR Fungal/Bacterial DNA MiniPrep™ was used to extract genomic DNA from B. neritina colonies for assessment of host genotype and symbiotic status by PCR. Analysis of the transcriptome suggests that symbiont-produced bryostatins are involved in reproduction of host B. neritina via PKC activation.
Epichloë myecelium genomic DNA isolated using the ZR Fungal/Bacterial DNA MiniPrep™ in a study identifying mutations in perA including a mutation in the C-terminal reductase domain resulted in some distinct species with nonfunctional peramine synthesis.
DNA was isolated from various Lactobacillus species using the ZR Fungal/Bacterial DNA MiniPrep™ to characterize and sequence the different strains in cheese. This allows for greater identification of the relationship between human diet and the intestinal microbiome.