The purified bacterial genomic DNA is ready for downstream applications such as:
- restriction endonuclease digestions
- Southern blots
- Starting material: Up to 1.5 mL of culture
- Expected yield: Up to 20 μg
- Elution volume: 400 μl
- Time required: 70 - 120 min
- A260/A280 ratio: 1.6 - 1.9
- No phenol, chloroform, or ethanol precipitation required
The GenElute Bacterial Genomic DNA kit provides a simple and convenient way to isolate pure genomic DNA from gram-negative bacteria. For most gram-positive bacteria, the kit must be used in conjunction with the optional lysozyme (L4919) to effectively lyse the thick peptidoglycan cell walls. A Gram-Positive Lysis Solution is provided as a diluent for preparing the lysozyme stock solutions.
The kit combines the advantages of a silica-based system with a microspin format and eliminates the need for expensive resins, alcohol precipitation, and hazardous organic compounds such as phenol and chloroform.
The bacteria are lysed in a chaotropic salt-containing solution to ensure the thorough denaturation of macromolecules. The addition of ethanol causes the DNA to bind when the lysate is spun through a silica membrane into a microcentrifuge tube. After washing to remove the contaminants, the DNA is eluted in 200 μL of a Tris-EDTA solution.
The expected yield of genomic DNA will vary depending on the cell density of the bacterial culture and the bacterial species and strain used. DNA purified with the GenElute kit has an A260/A280 ratio between 1.6 and 1.9 and can be up to 50 kb in length.