Preparation of Stock Solution of Ampicillin (50 mg/ml)

OVERVIEW

• Ampicillin is one of the most extensively used antibiotics as a selection marker in molecular biology and bacteriology. Many commonly used cloning and expression vectors have an ampicillin resistance gene.
• A 50 – 100 mg/ml stock solution of ampicillin is generally prepared in water. Depending on the need, the stock solution is diluted to the final working concentration.
• Commercially, ampicillin is available in three forms – Ampicillin anhydrous, Ampicillin trihydrate and Ampicillin sodium salt.
• Ampicillin anhydrous and Ampicillin trihydrate are only slightly soluble in water.
• The solubility of Ampicillin anhydrous is 10 mg/ml and Ampicillin trihydrate is 8 mg/ml in distilled water at 37°C (Hill et al., 1975).
• Ampicillin sodium salt is most often used to prepare stock solution as it has better solubility in water (>50 mg/ml in water).
• A 50 mg/ml stock solution of ampicillin can be prepared by dissolving 500 mg ampicillin sodium salt in water to a final volume of 10 ml.
  

REQUIREMENTS

Reagents
Ampicillin Sodium salt
Deionized / Milli-Q water

Ampicillin Sodium salt can be purchased from Sigma Aldrich (Catalog no. A9518) or ThermoFisher Scientific (Catalog number: 11593027) For more suppliers, Click here.

Equipment and disposables
15 ml graduated tube with screw-cap
Sterile Syringe Filter Unit containing a membrane with pore size 0.22 µm or below

Composition
50 mg/ml ampicillin

Objective
Preparation of 10 ml of 50 mg/ml ampicillin stock solution in water

PROCEDURE

Step 1: Dissolve 500 mg of Ampicillin Sodium in 9 ml of Milli Q water.
◊ Weigh out and transfer 500 mg of ampicillin to a 15 ml graduated screw-capped tube. 
◊ Add 9 ml of deionized / Milli-Q water. 
◊ Mix until all ampicillin is completely dissolved.

Tips:
1. You can mix by inverting the tube a number of times or using a rotator or vortexer.
2. Use autoclaved sterile deionized / Milli-Q water.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Cap the tube and mix by the solution inverting the tube a few times to make homogeneous solution.

Tip:
Centriguge the tube for a short time to bring all the liquid to the bottom. Now adjust the volume by adding water dropwise. Since graduated tubes have milliliter marks, you can easily adjust the volume without any need of transferring to measuring cylinder/volumetric flask.

Step 3: Sterilize the solution by filtering through a sterile membrane of porosity 0.22 µm or less under aseptic conditions.

Precaution:
Never sterilize the ampicillin solution by autoclaveing as it will degrade all ampicillin.

Storage
Stock solution can be stored at 4°C for a few days (1 week). Store the solution at -20°C for a long time (up to 1 year).

Tips:
1. It is recommended to store the ampicillin stock solution as small aliquots (0.5 – 1 ml) to protect the solution from repeated freeze-thaw cycles. The repeated freeze-thaw cycle can cause ampicillin degradation.
2. Aliquoting must be done under aseptic conditions.

Applications
Ampicillin is one of the most commonly used selection markers for bacterial culture during gene cloning experiments.

REFERENCES:

Hill et al., 1975. Dissolution and bioavailability of the anhydrate and trihydrate forms of ampicillin. J Pharm Pharmacol. 27(8), 594-8. PMID-239175