Antibiotic Stock Solutions

LAB PROTOCOL

Ampicillin

Preparation of 25ml stock solution of 100mg/ml (1000x stock)

1. Weigh 2.5g of ampicillin into a small weigh boat.

2. Add 12.5ml of milliQ H20 and 12.5ml of ethanol to a 50ml Falcon tube.

3. Add the ampicillin to the 50% ethanol mix.

4. Mix/vortex so all the ampicillin goes into solution.

5. Store at -20C.

Antibiotic Mode of Action: Inhibits the formation of cross-links in the peptidoglycan layer (which provides rigidity to the cell wall). Most effective against cells in log phase growth (since this is when new cross-links are being formed), and has little effect on cells in stationary phase.

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Mechanism of Resistance: Expression of β-lactamase neutralizes ampicillin. The resistance gene is named bla or ampR. When this enzyme is expressed on a high-copy number plasmid there is significant diffusion into the extracellular medium. As a result non-resistant satellite colonies may form around larger resistant colonies.

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Working Solution: Typical concentrations of ampicillin are 50 μg/mL for low copy plasmids and 100 μg/mL for high copy plasmids. Stock solutions are typically at 100 mg/ml, so that 1 ml of antibiotic can be added to 1 liter of broth or agar. Stock solutions made in 50% alcohol remain liquid at -20°C and are easy to pipet. Cool agar to 55°C or below prior to adding antibiotic.

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Chloramphenicol

Preparation of 25ml of 34mg/ml stock solution  (1000x)

1. Weight 0.85g of chloramphenicol sulfate into a small weight boat.

2. Add 25ml 100% EtOH to a 50ml Falcon tube and add chloramphenicol

3. Mix/vortex vigorously so all the chloramphenicol goes into solution.

4. Store at -20C.

Antibiotic Mode of Action: Chloramphenicol is a bacteriostatic agent that binds to the 50S ribosomal subunit and inhibits ribosomal peptide bond formation. It is sometimes used as a way of "amplifying" plasmid production by shutting down protein synthesis in cultures, while allowing plasmid replication to continue.

Working Concentrations: High copy plasmids allow 34 μg/ml concentration. Low-copy plasmids like 12.5 μg/ml.

Resistance Gene: The cat gene codes for an acetyltransferase which acetylates chloramphenicol, inactivating it.

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Kanamycin

Preparation of 25ml of 50mg/ml stock solution (1000x)

• Weigh 1.25g of kanamycin into a small weigh boat.

• Add 25ml of milliQ H20 to a 50ml Falcon tube and add kanamycin.

• Mix/vortex so all the kanamycin goes into solution.

• Filter sterilize the solution into a falcon tube using a 20ml syringe and a 0.22 μm filter.

• Aliquot into 1.5ml Eppendorf tubes.

Antibiotic Mode of Action: Bacteriocidal. Diffuses through the porin channels in the outer membrane of gram-negative bacteria. Interacts with at least three ribosomal proteins, inhibiting protein synthesis and increasing translation errors.

Mechanism of Resistance: Kanamycin is inactivated by bacterial aminophosphotransferases (APHs). The APHs inactivate kanamycin by transferring the γ-phosphate of ATP to the hydroxyl group in the 3' position of the pseudosaccharide. The KanR gene codes for kanamycin resistance. Several versions of this gene exist, with varying crossover resistance to other antibiotics such as neomycin or gentamycin.

Working Concentration and Stock Solution: Working concentration is 50 μg/ml, or 25 μg/ml for low-copy plasmids.