NEBExpress® T4 Lysozyme Lysis Protocol (NEB #P8115)

This protocol recommends using a Tris-based buffer to lyse E. coli. If using a detergent-based buffer or a buffer with EDTA, NaCl or MgCl2, refer to the table below for recommended usage guidelines.

  1. If monitoring cell density, record final OD600 readings prior to harvesting the bacterial cells.

  2. Harvest cells by centrifugation at 16,000 x g for 10 minutes at 4°C. For larger volumes, centrifuge for ≥30 minutes at ≤10,000 x g. Discard the medium and, if possible, weigh the wet cell pellet.

  3. Store the pellet at -20°C or -80°C or use immediately.

  4. Resuspend the cell pellet in 50 mM Tris-HCl pH 7.5 and mix until the suspension is homogenous:

    • Resuspend 1 UOD600 of cells in ≥ 0.1 mL of buffer.


      To calculate the UOD600, multiply the volume of cells harvested by the OD600 reading. For example, a 5 mL cell culture harvested at an OD600 of 2 gives 5 mL x 2 = 10 UOD600. 10 UOD600 x 0.1 mL buffer = 1 mL resuspension buffer.

    • If the wet cell pellet was weighed, resuspend 1 gram of cells in 30 mL of buffer.

  5. Add 1 µL of NEBExpress T4 Lysozyme per 1 mL of resuspended cells.

  6. Incubate the reaction at room temperature or 4°C for 5 minutes, with gentle shaking.

  7. If viscosity appears:

    1. Add 1 µL Micrococcal Nuclease (NEB #M0247S) per 1 mL of lysate with 1 mM CaCl2, mix and incubate for 5 minutes at room temperature.

      or

    2. Add 1 µL of DNase I (NEB #M0303S) per 1 mL of lysate with 1 mM CaCl2 or 1 mM MgCl2, mix and incubate for 5 minutes at room temperature.

      or

    3. Briefly sonicate the lysate with one or two pulses.

  8. Centrifuge the lysate at 16,000 x g for 10 minutes at 4°C to pellet the insoluble material (30 minutes or longer for large volumes and lower speed).

    Note: If the insoluble pellet looks fluffy, repeat step 7, followed by centrifugation.

  9. Carefully transfer the supernatant into a new container. This soluble fraction can be stored at 4°C for a few hours or -20°C for longer term storage.

  10. If needed, resuspend the insoluble pellet in 50 mM Tris-HCl pH 7.5 or any desired buffer for analysis or purification of inclusion bodies.

Resuspension buffer (1 mL) NEBExpress T4 Lysozyme Lysis efficiency Viscosity removal with
Micrococcal nuclease (NEB #M0247S)
NEBExpress E. coli Lysis Reagent (NEB #P8116) 0.1 µL Optimal Required
10-50 mM Tris-HCl pH 7.5, 0.5 mM EDTA or
5-40 mM HEPES pH 7.5, 0.5 mM EDTA
1 µL Very good If needed
10-50 mM Tris-HCl pH 7.5
or
5-40 mM HEPES pH 7.5
1 µL Good If needed
20-100 mM Sodium Phosphate pH 7.5 (+/- 0.5 mM EDTA) 1 µL Medium If needed
Buffer with >25 mM NaCl 1 µL Weak
(50 mM NaCl causes 80% inhibition)
If needed (inactive with >100 mM NaCl)
Buffer with >1 mM MgCl2 1 µL Weak
(5 mM MgCl2 causes 80% inhibition)
If needed

Notes:

  • If the lysis is performed with a denser cell suspension (cells resuspended in a ratio less than 0.1 mL per UOD600), NEBExpress T4 Lysozyme will lyse with less efficiency.

  • When working with non-E. coli strains, especially gram-positive bacteria, use 5 to 15 µL per 1 mL of resuspended cells to achieve lysis.

  • A sample of the harvested soluble fraction (supernatant) or resuspended insoluble pellet can be analyzed by SDS-PAGE. For 100 µL lysate from 1 UOD600 of cells, mix 20 µL of the sample with 10 µL of Blue Protein Loading Dye (NEB# B7703S). Incubate 2 minutes at 95°C, and load 10 to 20 µL per well.