{"product_id":"neb-p8116s","title":"New England Biolabs, P8116S, NEBExpress® E. coli Lysis Reagent","description":"NEBExpress  \u003cb\u003eRelated Categories\u003c\/b\u003e Competent Cells  \u003cb\u003eApplications\u003c\/b\u003e High-throughput cloning and automation solutions,, Protein Expression in, E. coli, ,, Protein Expression  \u003cb\u003eFAQ\u003c\/b\u003e Q: How much NEBExpress® E. coli Lysis Reagent should I use? A: We recommend using 5 mL of NEBExpress E. coli Lysis Reagent per 1 gram of cells. Alternatively, the volume of Lysis Reagent can be calculated based on the number of units OD600 (UOD600) harvested, where UOD600 is the OD600 of a culture multiplied by its total volume. For example, if a 5 mL culture is harvested when the OD600 is 1.0, 5 mL x 1.0 = 5 UOD600. It is recommended to use 0.025-0.075 mL of Lysis Reagent for every 1 UOD600 harvested, so in the example above 0.125 – 0.375 mL would be required. Q: Why do I see precipitation in the NEBExpress® E. coli Lysis Reagent? A: Use and\/or storage of the Lysis Reagent at temperatures \u0026lt; 4°C can cause the detergents to precipitate. To dissolve precipitates, simply allow the reagent to reach room temperature and mix. Q: Can I use NEBExpress® E. coli Lysis Reagent that has been frozen? A: Yes, allow it to come to room temperature and mix. Q: Should I chill the NEBExpress® E. coli Lysis Reagent prior to use? A: No, protein extraction is most efficient when the reagent is between 15 and 30°C (room temperature). Chilling the reagent prior to use will cause the detergents to precipitate, thereby resulting in incomplete protein extraction. Q: Should I freeze the cell pellets prior to lysing with NEBExpress® E. coli Lysis Reagent? A: Freezing is not necessary; however, frozen cell pellets may result in higher protein extraction efficiency. Q: What buffer is present in the NEBExpress® E. coli Lysis Reagent? A: The NEBExpress E. coli Lysis Reagent is Tris-based pH 7.5. Q: Can I supplement the NEBExpress® E. coli Lysis Reagent with NaCl? A: Yes, the NEBExpress® E. coli Lysis Reagent is compatible with NaCl concentrations ≤ 1M. Higher concentrations will cause precipitation. However, if used in the presence of NEBExpress® T4 Lysozyme (NEB #P8115), the NaCl concentration should be kept ≤25 mM. If used in the presence of Micrococcal Nuclease (NEB #M0247S) or DNase I (NEB #M0303), the NaCl concentration should be kept ≤100 mM. If a higher NaCl concentration is required, the NaCl concentration should be adjusted after performing the lysis step. Q: Can I change the pH of NEBExpress® E. coli Lysis Reagent? A: Yes, you can use a Tris-, PIPPS-, or Phosphate-based buffer to titrate the pH between 5.5 and 10. pH values lower than 5.5 will cause components of the reagent to precipitate. Q: Is the NEBExpress® E. coli Lysis Reagent compatible with EDTA? A: Yes, to prevent interference with metal affinity resins use \u0026lt; 0.1 mM EDTA. If using NEBExpress Ni Resin (NEB #S1428S) or Ni Spin Columns (NEB #S1427S\/L), no consideration for EDTA concentration is necessary, as the resin in these products is chemically resistant to EDTA chelation. If using NEBExpress® T4 Lysozyme (NEB #P8115) in association with NEBExpress® E. coli Lysis Reagent, we recommend using \u0026lt;2 mM EDTA. Q: Can I add a protease inhibitor to the NEBExpress® E. coli Lysis Reagent reaction? A: Yes, but choose a protease inhibitor compatible with any necessary downstream processing of the protein of interest. If using Factor Xa (NEB #P8010), Enterokinase (NEB #P8070) or Thrombin, avoid using serine protease inhibitors. If using TEV Protease (NEB #P8112), avoid using cysteine protease inhibitors If using metal-dependent enzymes and\/or proteases avoid EDTA\/EGTA. Q: Do I need to add lysozyme to aid in lysis? A: While not always necessary, the addition of lysozyme will improve extraction efficiency. Add 1 µL of NEBExpress® T4 Lysozyme (NEB #P8115) per 1 mL of lysate. The lysate may become viscous and require addition of 200-2000 U\/mL Micrococcal Nuclease (NEB #M0247S) or 10-100 U\/mL DNase I (NEB #M0303) in the presence of 1mM CaCl2. Q: My lysate is viscous; how can I resolve this? A: A viscous lysate results from an abundance of DNA released by the Lysis Reagent. Add 200-2000 U\/mL Micrococcal Nuclease (NEB #M0247S) or 10-100 U\/mL DNase I (NEB #M0303) in the presence of 1mM CaCl2. It’s recommended to perform a small-scale lysis test with the Lysis Reagent to determine whether Micrococcal Nuclease or DNase I are necessary followed by scale up. NEB’s DNase I is fused to maltose-binding protein (MBP), making its removal via amylose resin (NEB #E8021), amylose resin high flow (NEB #E8022), or amylose magnetic beads (NEB #E8035) simple and effective. Q: Can I use a reducing agent with NEBExpress® E. coli Lysis Reagent? A: Yes, NEBExpress E. coli Lysis Reagent is compatible with all commonly used reducing reagents, including β-mercaptoethanol, DTT, TCEP, and THP. For downstream purification, NEBExpress® Ni Spin Columns (NEB #S1427) and NEBExpress Ni Resin (NEB #S1428) are chemically resistant to these reducing reagents; immobilized metal affinity resins (e.g., Nickel or Cobalt) from other vendors may not be, so check with the supplier and choose an appropriate reducing agent. Q: Does NEBExpress® E. coli Lysis Reagent lyse gram-positive bacterial cells or yeast cells? A: NEBExpress® E. coli Lysis Reagent lyses some gram-positive bacteria such as certain Bacillus strains (B. subtilis, B. thuringiensis). Addition of NEBExpress® T4 Lysozyme (NEB #P8115) and Micrococcal Nuclease (NEB #M0247S) is highly recommended to obtain optimal lysis of gram-positive bacteria. NEBExpress® E. coli Lysis Reagent has limited activity on yeast cells. Q: Does NEBExpress® E. coli Lysis Reagent lyse gram-negative bacterial cells? A: Yes, we have tested and confirmed lysis of the following gram-negative bacteria: Vibrio natriegens, Salmonella typhimurium, Pseudomonas putida, Xanthomonas malvacearum, Ralstonia eutropha, Flavobacterium johnsoniae and all E. coli expression strains available in the NEB catalog. Q: My protein forms inclusion bodies. Can I still use this reagent to lyse my cells? A: Yes, and NEBExpress® E. coli Lysis Reagent will help clear up inclusion bodies. Q: Can I add NEBExpress® E. coli Lysis Reagent directly to my cell culture to lyse the cells? A: Yes, we recommend using a 1:1 ratio of Lysis Reagent to cell culture or to washed and concentrated cells in a desired buffer as long as the buffer is compatible with downstream steps. NEBExpress® T4 Lysozyme (NEB #P8115) and Micrococcal Nuclease (NEB #M0247S), may also be added if needed. 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