Reducing viscosity in an E. coli lysate with NEBExpress® Salt Active Nuclease (NEB #M0764)
- Resuspend 1 g of wet E. coli cell pellet in 5 mL of either 50 mM Tris-HCl, 0.1 mM EDTA, pH 8.5 or NEBExpress E. coli Lysis Reagent (NEB #P8116).
- Add 5 µL of NEBExpress T4 Lysozyme (NEB # P8115) and incubate at 4°C for 15 minutes, with gentle mixing. A viscous lysate should appear.
- Add 500 µL of 5 M NaCl (500 mM NaCl final concentration) and gently mix.
- Dilute NEBExpress Salt Active Nuclease 10-fold to a final concentration of 10 U/µL by combining 2 µL Salt Active Nuclease and 18 µL 50 mM Tris-HCl, 500 mM NaCl, 5 mM MgCl2, pH 8.5.
- Add 5 µL of diluted NEBExpress Salt Active Nuclease (equivalent to 10 U/mL of reaction).
- Incubate for 10 minutes at room temperature (RT). The lysate is now ready for further processing.
Note: Optimal enzyme concentration and incubation times may vary depending on the sample preparation (pH, salt concentration, DNA and RNA concentration), the temperature of reaction (typically between 4°C and 37°C) and the application.