Prepare Bacterial Lysates by Sonication

Getting Started

You have bacterial cultures and you want to extract something from inside of them (e.g., expressed protein from an expression strain, genomic DNA, total lysate, etc.). You’re going to need to lyse this culture. This protocol shows you how to use a sonicator to mechanically lyse cells with ultrasonic blasts.

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, cooling block, and lysis buffer.

Prechill centrifuge and lysis buffer to 4C.

Prechill centrifuge tube cooling block in -20C freezer.

Finish lysis buffer with TCEP (500x, or 1 mL TCEP per 2 L buffer).

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Notes - keep lysate cold to prevent proteolysis!

Resuspend pellets in cold lysis buffer.

Thaw bacterial pellets on ice.

Add 10 mL lysis buffer to each pellet. Resuspend pellets completely by vortexing.

Transfer resuspended pellets to 15 mL centrifuge tubes

Lyse cells by sonication.

Set sonicator to appropriate setting. We lysed cells to a final energy of 12 000J / 40 mL total lysate (50% amplitude, 20s on / 20s s off).

Put resuspended pellets in an ice bucket. Place cooling block on lab jack beneath the sonicator tip(s).

Adjust sample height using the lab jack such that the sonicator tip(s) are submerged about one quarter (1/4) of the way into the sample (e.g., 2.5 mL into a 10 mL sample).

Lyse cells by sonication.

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Notes - always use a Sound Enclosure during sonication

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Notes - avoid foam during sonication!

Clarify lysates by centrifugation and filtration.

Centrifuge lysates at 4 krcf / 4C / 45 min.

(Meanwhile) for each lysate, assemble 10 mL Luer lock syringes and filters (we used 0.45 um for lysates). Pull the plunger out of the syringe, screw on the filter, and leave the plunger outside of the syringe.

For each lysate, decant the supernatant into syringe and put in the plunger. Plunge lysate through filter into a collection tube (we used 15 mL centrifuge tubes).

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Notes - keep going! Don’t take a break!