Overview
- The 2X YT medium is also known as a 2 X TY medium.
- It is a rich growth medium optimized for growth and maintenance of filamentous phage such as M13
- It is recommended for the growth and propagation of E. coli infected with the single strand fillamentous bacteriophage M13.
- It contains all the nutritional requirements for E.coli
- It contains tryptone, yeast extract, and sodium chloride.
- Tryptone is a pancreatic digest of casein (also called Peptone C). It functions as an amino acid source for microorganisms.
- Vitamins and certain trace elements are provided by yeast extract.
- Sodium chloride (NaCl) supplies essential electrolytes for transport and osmotic balance.
- Many supplements including antibiotics, are heat-sensitive and cannot be autoclaved. These should be filter-sterilized and added to the medium after it has cooled down.
Reagents Required
- Yeast extract
- Tryptone
- Sodium chloride (NaCl)
- 5N NaOH (for pH adjustment)
- Deionized / Milli-Q waterE
Equipment
- Measuring cylinder
- Conical flask / Beaker
- Hot plate / Magnetic stirrer with a heating device
Preparation
Step 1: To prepare 1000 ml of 2 X YT medium, weigh out 16 g tryptone, 10 g yeast extract, 5 g Sodium chloride (NaCl) in a 2-liter conical flask/beaker. Add 800 ml deionized/Milli-Q water and mix it. The solution will appear translucent-yellowish with undissolved media ingredients.
Tip:
One can use manual shaking using a glass pipette to mix the ingredients. The magnetic stirrer makes the dissolving process easy and convenient.
Precautions: 1. Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in the solvent. 2. Make sure no lumps are remaining after making the suspension of medium components.
Step 2: Dissolve all ingredients completely by heating to boiling while stirring.
Note:
Medium with completely dissolved ingredients will appear as a transparent yellowish solution.
Precautions: 1. Once all the ingredients of the medium are dissolved and the medium appears transparent, stop the heating. Don’t unnecessarily heat the medium. 2. Make sure to dissolve any residual powder sticking to the glass.
Step 3: Cool down the medium to room temperature. Adjust the pH 7.0 with 5N NaOH (~0.2 ml).
Note:
The 2 X YT medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches near the saturation phase.
Precaution:
Since pH is dependent on temperature, It is always a good practice to adjust the medium pH only after the solution has cooled to 25 °C (room temperature).
Step 4: Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.
Step 5: Transfer the medium to an autoclavable bottle or cover the conical flask with a cotton plug and aluminum foil.
Tip:
One can make small aliquots of the medium if needed (e.g., 5 ml aliquots in 50 ml conical flask).
Step 6: Sterilize the solution by autoclaving 20 minutes at 15 lb/sq.in. (psi) from121-124°C on liquid cycle).
Precautions: 1. Antibiotics and nutritional supplements should be added only after the solution has cooled to 40°C or below. Many supplements, like antibiotics, are degraded at high temperatures. 2. After autoclaving, all sterile solutions should be handled inside the laminarow hood to maintain sterility.
Storage
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage.
Preparation for other volumes
| 100 mL | 500 mL | 1000 mL | 10000 mL | |
| Bacto-tryptone | 1.6 g | 8 g | 16 g | 160 g |
| Bacto-yeast extract | 1 g | 5 g | 10 g | 100 g |
| Sodium chloride | 0.5 g | 2.5 g | 5 g | 50 g |
| Water | to 100 mL | to 500 mL | to 1000 mL | to 10000 mL |