I want to study the LC-MS analysis of a bacterial metabolite.The bacteria is a carotenoid producing bacteria. I have added methanol to the cell pellet and then undergo sonication for cell lysis.After centrifugation I have taken the supernatant.How do I remove nucleic acid from supernatant to isolate metabolites?
Hello Sanhita Saha
You may add Benzonase Nuclease to the cells prior to lysis. Benzonase Nuclease is an endonuclease that attacks and degrades all forms of DNA and RNA (single stranded, double stranded, linear and circular). Benzonase Nuclease is ideal for a wide variety of applications where complete digestion of nucleic acids is desirable.
FYI: Benzonase Nuclease requires 1‒2 mM Mg2+ for activity. Benzonase is inhibited (approximately 50% activity) by monovalent cation concentrations >50%,
phosphate concentrations >20 mM, and by ammonium sulfate concentrations >25 mM.
You can achieve reversible inhibition using EDTA to chelate essential metal ions, and irreversible inactivation can be accomplished using 100 mM NaOH at 70 °C for 30 minutes.
The optimization of conditions will depend on reaction time, temperature, and concentration as these three factors influence each other. Preferably, use Tris buffer and temperature at 37 degree C.
Alternatively, you may use DNase I and RNase A to degrade DNA and RNA respectively in the supernatant.
Best.
You could run your lysate through a nucleic acid purification column and continue with the flow through for the MS analysis. Alternatively, If ethanol or isopropanol don't harm, use them to either precipitate the nucleic acids (or evaporate the methanol and extract the residue with them) or directly use these alcohols to extract your molecules (should be easy to follow visibly) while the nuclei acids won't be solubilized.
- Badges
- Science topic
- Similar topics
- Filing