Staphylococcal nuclease (Staph Nuc) is a heat-stable, potent secretory protein that cleaves dsDNA in vitro. You have a cloned the Staph Nuc gene on pBR322 plasmid and transferred into E. coli. You have named the chimeric plasmid pBR322 with the cloned Staph nuc gene as pNuc. In an experiment, it was necessary that you purify pNuc from an E. coli culture, but was unsuccessful. The cause for being unable to purify the pNuc plasmid was that the Staphylococcus nuclease enzyme hydrolyzing the plasmid along with the chromosomal DNA immediately after cell lysis and released in the salt buffer during purification process. How would solve this issue and protect the pNuc plasmid DNA so that you can proceed with the downstream molecular analyses of the gene?
To protect the pNuc plasmid DNA from hydrolysis by the Staphylococcus nuclease enzyme, you can employ a few strategies during the purification process. Here's how you can solve this issue and ensure the integrity of the pNuc plasmid DNA for downstream molecular analyses:
1. Modify the lysis conditions: Optimize the lysis conditions to minimize the release of endogenous nucleases from the E. coli cells. You can adjust the pH and temperature of the lysis buffer, or include a nuclease inhibitor such as EDTA or phenylmethylsulfonyl fluoride (PMSF) to deactivate the nucleases.
2. Use a nuclease inhibitor cocktail: Rather than using a single inhibitor, you can use a combination of multiple nuclease inhibitors to ensure effective protection against different classes of nucleases. Common inhibitor cocktails, such as the "Complete" cocktail from Roche, contain a broad spectrum of protease and nuclease inhibitors.
3. Quick homogenization and immediate cooling: Perform cell lysis quickly and efficiently to minimize exposure to nucleases. After cell lysis, transfer the lysate to an ice bath or use a cooling device to rapidly reduce the temperature, as most nucleases are temperature-sensitive.
4. Include a chelating agent in the purification buffer: Add a chelating agent like EDTA to the purification buffer. EDTA chelates divalent metal ions (such as Mg2+ and Ca2+), which are often required for nuclease activity. By sequestering these ions, you can inhibit nuclease activity and protect the plasmid DNA.
5. DNAse inactivation: Inactivate the nuclease enzyme by denaturing or removing it. You can heat-treat the lysate at a high temperature (e.g., 70-80°C) for a short duration to denature the nucleases. Alternatively, you can use affinity chromatography techniques to capture and remove the nuclease enzyme.
6. Optimize purification methods: Choose a purification method that minimizes exposure of the DNA to enzymes. For example, you can consider using DNA extraction kits based on silica-binding or column-based purification methods. These methods often involve binding the DNA to a solid support, washing away contaminants, and then eluting the purified DNA under controlled conditions.
By implementing these measures, you can protect the pNuc plasmid DNA from degradation and ensure its integrity for downstream molecular analyses.