Getting Started
You have bacterial cultures and you want to extract something inside of them (e.g., overexpressed protein, plasmid DNA, tRNAs, total lysate, etc.). You’re going to need to lyse this culture. You can use an sonicator to do so, disrupting bacterial walls and membranes using ultrasonic frequencies pumped into the bacterial culture. This protocol shows you how to use a sonicator to mechanically lyse cells.
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
Sonicator | ||||||
Sonicator | Q700 Sonicator | QSonica | Q700A-110 | $5500 | 4C to 30C | [link] |
Sonicator Probe | Standard Probe, Replaceable Tip, 1/2” (12.7mm) diameter | QSonica | 4220 | $670 | 4C to 30C | [link] |
Sound Enclosure | Sound Enclosure | QSonica | 432B2 | $995 | 4C to 30C | [link] |
Lab Jack | Fisherbrand™ Lab Jacks (15cmx15cm) | Fisher Scientific | 14-673-51 | $484 | 4C to 30C | [link] |
Consumables | ||||||
15 mL centrifuge tubes | Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case | Corning | 430790 | $230.44 | 4C to 30C | [link] |
10 mL Luer Lock Syringes | Syringes with BD Luer-Lok® Tip, BD Medical, Syringe with Luer-Lok® Tip, Volume=10 mL | BD Industrial/Difco | 550-80620-PK | $102.40 | 4C to 30C | [link] |
0.45 um syringe filters | PES Syringe Filter, 0.45μm, 30mm, Sterile | CELLTREAT Scientific Products | 229749 | $84 | 4C to 30C | [link] |
Salts (for Buffers) | ||||||
Potassium Hydroxide | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4C to 30C | [link] |
HEPES | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4C to 30C | [link] |
Ammonium Chloride | Ammonium chloride,ACS reagent, ≥99.5% | Sigma-Aldrich | 213330-500G | $73.50 | 4C to 30C | [link] |
Sodium Chloride | Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99% | Sigma-Aldrich | 746398-1KG | $133.00 | 4C to 30C | [link] |
Magnesium Chloride hexahydrate | BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | 4C to 30C | [link] |
Supplements (for Buffers) | ||||||
Lysozyme | Lysozyme from chicken egg white, protein ≥90%, ≥40,000 units/mg protein, lyophilized powder | Sigma-Aldrich | L6876-1G | $76.90 | -25C to -15C | [link] |
TCEP-HCl (0.5M; presuspended) | Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL | Thermo Scientific | 77720 | $175.65 | 4C to 30C | [link] |
cOmplete Protease Inhibitor | cOmplete™ Protease Inhibitor Cocktail, EDTA-Free, Tablets | Roche | 11873580001 | $472 | 1C to 4C | [link] |
Prerequisite Protocols
Prepare lysis buffer:
Make Protein Purification Buffers and Media.
Prepare pellets of bacteria expression proteins:
Grow and Induce Expression Strains.
Protocol
Prepare Stock Solutions.
Reagent | MW [g/mol] | Amount [g] | Final Volume [mL] | Storage | Needs pH adjustment? | Needs Sterilization? |
Stock Solutions | ||||||
Potassium Hydroxide (1M) | 56.11 | 14.0 | 250 | 4C to 30C | no | no |
HEPES-KOH (pH=7.6; 1M) | 238.3 | 59.5 | 250 | 4C to 30C; dark | yes | no |
Ammonium Chloride (1M) | 53.49 | 13.4 | 250 | 4C to 30C | no | no |
Sodium Chloride (5M) | 58.44 | 73.1 | 250 | 4C to 30C | no | no |
Magnesium Chloride (1M) | 203.3 | 20.3 | 100 | 4C to 30C | no | no |
Lysozyme (30 mg/mL) | 14320 | 1 | 33 | -25C to -15C | no | no |
Prepare Lysis Buffer - used to resuspend bacterial pellets for mechanical lysis by sonication. Also chemically lyses cells with lysozyme (G. gallus). Contains protease inhibitor (cOmplete) to slow proteolysis. Make fresh and use the same day!
Reagent | Final Concentration [mM] | Stock Concentration [mM] | Volume to Add [mL] |
HEPES-KOH | 50 | 1000 | 5 |
Ammonium Chloride | 100 | 1000 | 10 |
Sodium Chloride | 500 | 5000 | 10 |
Magnesium Chloride | 10 | 1000 | 1 |
Lysozyme | 0.3 mg/mL | 30 mg/mL | 1 |
TCEP | 1 | 500 | 0.2 |
cOmplete Protease Inhibitor | 1 tablet / 50 mL | n/a | 2 tablets |
water (milliQ) | 72.8 | ||
Total | 100 |
Prepare centrifuge and Lysis Buffer.
Pre-chill your centrifuge and Lysis Buffer to 4C.
Finish Lysis Buffer with TCEP (0.5M) (e.g., add 500 uL TCEP to 1 L Lysis Buffer).
Resuspend pellets in cold Lysis Buffer.
Thaw bacterial pellets on ice.
Weigh 1 g dry cell mass per pellet.
Resuspend 1 g pellets in 10 mL Lysis Buffer by vortexing.
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Lyse cells by sonication.
Program your sonicator. We recommend 50% amplitude, 10s on / 10s off.
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Place resuspended samples under the sonicator horn in an ice bucket on a lab jack.
Adjust your sample height using the sample jack until each sonicator tip is submerged by about one quarter (1/4) of the sample height (e.g., 2.5 mL into a 10 mL sample).
Lyse cells by sonication. Deliver 4.5 kJ for every 10 mL sample (e.g., a four-pronged sonicator horn lysing 4x 10 mL samples → 18 kJ).
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Clarify lysates by centrifugation and filtration.
Centrifuge lysates at 4 krcf / 4C / 45 min.
(Meanwhile) for each lysate, assemble a 10 mL Luer lock syringe and 0.22 um syringe filter.
Carefully decant each supernatant into a syringe and filter into a fresh 15 mL centrifuge tube. Keep clarified lysate cold (4C).
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Resources and References
- Papers
- Original PURE paper
- OnePot PURE
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Developers