About ZR-96 Soil Microbe DNA Kit™
The ZR-96 Soil Microbe DNA Kit™ is designed for the simple and rapid isolation of humic-free, PCR-quality DNA from microbes in soil. This product can be used to isolate DNA from tough-to-lyse bacteria, fungi, protozoa, and algae that inhabit a variety of samples including clay, sandy, silty, peaty, chalky, and loamy soils. Soil microbes are rapidly and efficiently lysed by bead beating with our state of the art, ultra-high density BashingBeads™. Fast-Spin plate technology is then used to isolate the DNA, which is subsequently filtered to remove humic acids/polyphenols that can inhibit PCR. The procedure can be performed in minutes, and there is no need for organic denaturants or proteinases.
|DNA Recovery||Typically, up to 5 µg total DNA (per well) is eluted into ≥ 25 µl DNA Elution Buffer per sample.|
|Processing Time||50 min|
|Equipment||Microcentrifuge, vortex, cell disruptor/pulverizer (optional).|
|DNA Size Limits||Capable of recovering DNA 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||DNA is isolated from bacteria, fungi, protozoa, and algae in up to 135mg of soil or from fungal/bacterial cells directly.|
|DNA Purity||High-quality, humic-free DNA is eluted with DNA Elution Buffer and is especially well suited for PCR (A260/280 nm >1.8.|
Metagenomic DNA isolated from 5 soil samples. M: 1 kb marker (NEB); 1-5: soil samples (sand, sandy clay loam, hydrophobic sandy loam, course sandy loam, fine gravel).
Add up to 135 milligrams of soil sample to the tubes of a ZR BashingBead™ Lysis Rack. Add 400 µl Lysis Solution to each tube. Cap tubes tightly to prevent leakage.
Alternatively, add 10-20 mg (wet weight) fungal and/or bacterial cells that have been resuspended in up to 50 µl of water or isotonic buffer (e.g., PBS) to the tubes of a ZR BashingBead™ Lysis Rack.
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 cell disrupters (e.g., 2000 GenoGrinder®, page 5). See manufacturer’s literature for 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 Soil DNA Binding Buffer to the supernatant in the 96-Well Block from Step 4. Cover completely with Cover Foil and mix thoroughly by vortexing 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 Soil 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 onto a clean Elution Plate and add 100 µl (50 µl minimum) DNA Elution Buffer directly to the matrices in the plate. Centrifuge the assembly at ≥ 3,000 x g for 5 minutes.
If fungi or bacterial cultures were sampled, the DNA is now suitable for PCR as well as other downstream applications.
Transfer the eluted DNA from Step 10 to a prepared Silicon-A™-HRC Plate mounted onto a clean Elution Plate. Centrifuge the assembly at exactly 3,500 x g for 3 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.