About Quick-DNA™ Fungal/Bacterial 96 Kit
Note: The ZR-96 Fungal/Bacterial DNA Kit™ (D6006) has been updated to Quick-DNA™ Fungal/Bacterial 96 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 96 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 plate 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 5 µg total DNA is eluted into ≥ 25 µl DNA Elution Buffer per sample.|
|Processing Time||40 min|
|Equipment||Centrifuge w/ microplate carriers, 96-well plate/block disruptor or pulverizer.|
|DNA Size Limits||Capable of recovering genomic DNA sized fragments from 100 bp to ≥40 kb. Typical fragment sizes range from 25 kb - 35 kb. If present, parasitic and viral DNA will also be recovered.|
|Sample Sources||10-20 mg (wet weight) fungi or bacteria. This equates to approximately 2x108 bacterial cells, 2x107 yeast cells and 2x106 mammalian cells. Spores, pollen, nematodes, other microorganisms, and up to 40 mg soft tissue (e.g., mammalian) can also be sampled.|
|DNA Purity||High quality DNA is eluted with DNA Elution Buffer making it perfect for PCR (A260/A280 nm >1.8.|
Fungal and bacterial DNA purification. DNA isolated from Saccharomyces cerevisiae (spores) and E. Coli using the Quick-DNA™ Fungal/Bacterial 96 Kit is high quality and structurally intact. Equivalent amounts of yeast and bacteria were processed using the Quick-DNA™ Fungal/Bacterial 96 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 10-20 mg (wet weight) fungal or bacterial cells that have been resuspended in up to 50 µl of water or isotonic buffer (e.g., PBS) or up to 40 mg of soft tissue (e.g., mammalian) to the tubes of a ZR BashingBead™ Lysis Rack. Add 400 µl Lysis Solution to each tube. Cap tubes tightly to prevent leakage.
Secure in a 96-well block/plate bead beater (e.g., 2010 GenoGrinder®) and process samples. Optimization of processing time/speed will be necessary for complete sample lysis.
Processing times may be as little as one minute when using high-speed bead beaters (e.g., 2000 GenoGrinderâ, page 4). See manufacturer’s literature for specific operating information.
Centrifuge the ZR BashingBead™ Lysis Rack at ≥ 3,000 x g (5,000 x g max.) for 5 minutes.
Transfer up to 250 µl supernatant to each well of a 96-Well Block.
Add 750 µl of Fungal/Bacterial DNA Binding Buffer to the supernatant in the 96-Well Block from Step 4. Cover completely with Cover Foil and mix thoroughly by vortexing block for 2 minutes. Centrifuge the 96-Well Block at ≥ 3,000 x g (5,000 x g max.) for 5 minutes.
Remove or pierce foil and transfer 500 µl of each of the supernatants from Step 5 to the wells of a Silicon-A™ Plate on a Collection Plate. Centrifuge the assembly at ≥ 3,000 x g (5,000 x g max.) for 5 minutes.
Discard the flow through from the Collection Plate and repeat Step 6.
Add 200 µl DNA Pre-Wash Buffer to the wells of the Silicon-A™ Plate on the emptied Collection Plate and centrifuge the assembly at ≥ 3,000 x g for 5 minutes.
Add 500 µl Fungal/Bacterial DNA Wash Buffer to the wells of the Silicon-A™ Plate on the Collection Plate and centrifuge the assembly at ≥ 3,000 x g for 5 minutes.
Transfer the Silicon-A™ Plate to an Elution Plate and add 100 µl (25 µl minimum) DNA Elution Buffer directly to the matrices in the plate. Centrifuge the assembly at ≥ 3,000 x g for 5 minutes.
Eluted, ultra-pure DNA is now ready for use in your experiments, or the Elution Plate can be covered with Cover Foil for storage of the DNA.