Preparation of Stock Solution of L-Glutamine (200 mM)

Overview

• L-Glutamine (C₅H₁₀N₂O₃; CAS number: 56-85-9) is an important component of the cell culture medium. It is an alternative energy source especially for the cell types with high metabolic demands (e.g., fast-growing cell lines) and consumes glucose inefficiently. In addition, it is also a source of nitrogen and carbon for the synthesis of cell components including nucleic acid and proteins.
• It is unstable in solution, therefore it is recommended to add L-glutamine in basal medium just prior to use. The L-Glutamine supplemented medium can be stored at 4 °C just for 2 weeks.
  
• The concentration of L-Glutamine may vary depending on cell types and culture medium. However, for most classical serum-grown cell lines, a 2 mM concentration is generally used. 
• As compared to other amino acids, a relatively high concentration of L-glutamine is used in cell culture medium (vary from 2 to 10 mM) as it is a growth-limiting amino acid for the growth of most cell types.
• It is soluble in water (solubility in water at 25 °C: 36 mg/ml) and yields a clear colorless solution.
• It degrades in the culture medium that results the accumulation of toxic of ammona in the culture medium.
  
• Since autoclaving degrades the glutamine solution, filter sterilization (filtering through a sterile membrane with pore size 0.22 micron or less) is used to sterilize L-glutamine solution.

Requirements

Reagents
> L-Glutamine (Molecular weight: 146.1456)
> Cell culture grade water (Milli-Q water) or 0.85% saline solution

Equipment and disposables
> Conical flask / Beaker (capasity 250 ml)
> Measuring cylinder
> Magnetic stirrer/glass rod

Composition:
200 mM L-Glutamine in water or 0.85% saline

Objective
Preparation of 100 ml of 200 mM L-Glutamine solution in water or 0.85% saline solution
Note: Glutamine solution can be prepared either in water or in 0.85% saline.

Preparation

Step 1: To prepare 100 ml of 200 mM L-Glutamine solution, dissolve 2.92 g L-Glutamine in 90 ml cell culture grade water or 0.85% saline.
> Weigh out 2.92 g of Glutamine and transfer it to a container (beaker/conical flask, capacity 250 ml) containing 90 ml water or 0.85% saline.
> Stir the solution using a glass rod/magnetic stirrer until all L-Glutamine dissolves completely.

Tip:
> Capacity of the container (beaker/conical flask) must be at least double the volume of the solution you intended to prepare. It helps to prepare the solution without spilling the solution out of the container.

Precaution:
> Make sure no undissolved L-Glutamine in the beaker.

Step 2: Adjust the volume to 100 ml with the solvent you used for the solution preparation (cell culture grade water or 0.85% saline).
> Transfer the solution to a volumetric flask or measuring cylinder. Adjust the final volume of 100 ml with the solvent. 
> After adjusting the volume 100 ml, transfer the solution back to the same container.

Step 3: Filter sterilize the solution
> Sterilize the solution by filtering it through a sterilized filter unit containing a filter paper with pore size 0.22 µm or less.

Precaution
The solution can not be autoclaved as autoclaving will destroy the L-Glutamine.

Storage:
Store the solution in small aliquots (5 ml) in the freezer (-20 °C). The solution is stable at -20C for several months.

Use Calculator to calculate the quantities of NaOH amount for the preparation of NaOH solution of specific concentration and volume

Molecular weight of NaOH: 40 g/mole