About Quick-gDNA™ MidiPrep
The Quick-gDNA™ MidiPrep is a simple procedure for the rapid isolation of total DNA (e.g., genomic, mitochondrial, viral) from a variety of biological sample sources. This product has been optimized for maximal recovery of ultra-pure DNA without RNA contamination and is compatible with whole blood (fresh or stored), serum, plasma, buffy coat, cells from culture, and many biological liquid samples.
For processing, simply add the specially formulated Genomic Lysis Buffer to a sample, vortex, and transfer the mixture to the supplied Zymo-Spin™ Column w/ Zymo-Midi Filter™. There is no need for organic denaturants or Proteinase K digestion because of the unique lysis buffer system. The product features Zymo-Spin™ technology to yield high-quality, purified DNA in just 20 minutes (see below). PCR inhibitors are effectively removed during the purification process. DNA purified using the Quick-gDNA™ MidiPrep is suitable for PCR, nucleotide blotting, DNA sequencing, restriction endonuclease digestion, bisulfite conversion/methylation analysis, and other downstream applications.
|DNA Recovery||Up to 125 µg total DNA is eluted into ≥150 µl DNA Elution Buffer or water. Human whole blood will typically yield 3-7 µg DNA per 100 µl blood sampled. Mammalian tissues already homogenized yield: 1-3 µg DNA per mg skeletal, heart, and brain tissues and 3-5 µg DNA per mg liver, kidney and lung tissues.|
|Equipment||Centrifuge or vacuum source and manifold, microcentrifuge, vortex.|
|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||Up to 3 ml (see protocols) whole blood, plasma, or serum from humans, mice, rats, etc. Also, cells from culture as well as a variety of biological liquids are effectively processed using this kit. Tissue already digested with Proteinase K or mechanically homogenized can also be processed.|
|DNA Purity||High-quality DNA is eluted with DNA Elution Buffer or water. DNA is especially well suited for PCR and other downstream applications. A260/A280>1.8|
|Product Detergent Tolerance||≤5% Triton X-100, ≤5% Tween-20, ≤5% Sarkosyl, ≤0.1% SDS.|
|Workflow Overview||Unique lysis buffer system omits the need for Proteinase K digestion for biological fluids and cell culture samples.|
Add 12 ml of Genomic Lysis Buffer to 3 ml1 (4:1) of blood, serum, or plasma. Mix completely by vortexing 4-6 seconds, then let stand 5 minutes at room temperature.
Note: Add 12 ml Genomic Lysis Buffer to all samples < 3 ml.
Transfer the mixture to a Zymo-Spin™ V-E Column/Zymo-Midi Filter™ assembly in a 50 ml tube2. Centrifuge the tube at ≥1,000 x g (2,000 x g max.) for 5 minutes3.
Note: If using a vacuum manifold, the processing capacity is reduced to 2 ml of blood, serum, or plasma + 12 ml Genomic Lysis Buffer per prep. This filtration step may take up to twenty minutes when using vacuum.
Disconnect the Zymo-Spin™ V-E Column/Zymo-Midi Filter™ assembly and transfer the Zymo-Spin™ V-E Column to a Collection Tube. Spin at 10,000 x g for 1 minute in a microcentrifuge4 to remove residue from the column.
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 of g-DNA Wash Buffer to the column and centrifuge at 10,000 x g for one 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 matrix5 and allow column to stand for 1 minute at room temperature. Centrifuge at 10,000 x g for 1 minute to elute the DNA6. The eluted DNA can be used immediately for molecular based applications or stored ≤-20oC for future use.