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.
- If monitoring cell density, record final OD600 readings prior to harvesting the bacterial cells.
- 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.
- Store the pellet at -20°C or -80°C or use immediately.
- 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.
- Resuspend 1 UOD600 of cells in ≥ 0.1 mL of buffer.
- Add 1 µL of NEBExpress T4 Lysozyme per 1 mL of resuspended cells.
- Incubate the reaction at room temperature or 4°C for 5 minutes, with gentle shaking.
- If viscosity appears:
- 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
- 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
- Briefly sonicate the lysate with one or two pulses.
- 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.
- 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.
- 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.
- 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.
