About Quick-DNA™ Fungal/Bacterial Midiprep Kit
Note: The ZR Fungal/Bacterial DNA MidiPrep™ (D6105) has been updated to Quick-DNA™ Fungal/Bacterial 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™ Fungal/Bacterial Midiprep 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 ~125 µg total DNA is eluted into ≥ 150 µl DNA Elution Buffer per sample.|
|Processing Time||20 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||150-250 mg (wet weight) fungi or bacteria or up to 500 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. A[260nm/280nm] >1.8, A[260nm/230nm] >2.0|
Fungal and bacterial DNA purification. DNA isolated from Saccharomyces cerevisiae (spores) and E. Coli using the Quick-DNA™ Fungal/Bacterial Midiprep Kit is high quality and structurally intact. Equivalent amounts of yeast and bacteria were processed using the Quick-DNA™ Fungal/Bacterial Midiprep 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 250-500 mg (wet weight) fungal or bacterial cells that have been resuspended in up to 1 ml of water or isotonic buffer (e.g., PBS) or up to 1 g of tissue to a ZR BashingBead™ Lysis/Filtration Tube1. Add 6 ml Fungal/Bacterial DNA Binding Buffer to the sample, cap tube2, 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 5) 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 5). 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 manifold3,4 (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 microcentrifuge5, 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 Fungal/Bacterial 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 matrix6 and allow column to stand for 1 minute at room temperature. Centrifuge at 10,000 x g for 1 minute to elute the DNA.
Ultra-pure DNA is now ready for use in your experiments.