2x YT medium
Introduction
2xYT medium is a nutritionally rich liquid formulation that is used for the propagation of recombinant strains of Escherichia coli. It is the recommended medium for the culturing of E.coli strains infected with M13 bacteriophage or other filamentous single-stranded DNA bacteriophages.
Principle
2x YT medium consists mainly of a tryptic digest of casein (tryptone), yeast extract, and sodium chloride. tryptone act as a source of nitrogenous compounds like amino acids and peptides. Yeast extract supplies the medium with B vitamins and minerals. Sodium chloride maintains the isotonicity of the medium. Together, these components provide the necessary growth factors and conditions that allow for rapid growth and replication of bacteriophages (without weakening the host cells). 2x YT medium also facilitates better recovery of and E.scherichia coli following heat shock during transformation.
Applications
- Used for culturing of Agrobacterium tumefaciens and E. coli DH10B.
- Non-selective cultivation of M13 bacteriophage, and E. coli strains for genetic research (cloning and production of DNA plasmids and recombinant proteins).
- For the growth of hosts for replication of M13 vectors without getting devitalized for a longer period.
- Acts as a selective medium when appropriate antibiotics or additional nutritional supplements are added.
Composition
| Ingredients | For 100mL | For 500mL | For 1000ml |
|---|---|---|---|
| Tryptone | 1.6 gm | 8 gm | 16 gm |
| Yeast extract | 1 gm | 5 gm | 10 gm |
| NaCl | 0.5 gm | 2.5gm | 5 gm |
| Distilled water | Upto 100 ml | Upto 500ml | Upto 1000ml |
Table 1. Composition of 2xYT medium.
pH
The pH of 2x YT medium is maintained at 6.8 ± 0.2 at 25°C; and is adjusted using NaOH and HCl.
Preparation
- Weigh the ingredients separately with respect to the volume of the media. (Here, we are considering 1L of the media).
- Suspend the ingredients in a glass beaker containing about 900mL of distilled water.
- Dissolve the components in the beaker using a magnetic stirrer. You may heat the solution to dissolve the components completely.
- Adjust the pH of the medium to the desired value.
- Makeup the volume to 1000mL.
- Transfer the broth to a conical flask or aliquot into smaller volumes.
- Close the mouth of the flask with a cotton plug; wrap the plug and neck of the flask with a piece of paper or aluminium foil.
- Sterilize by autoclaving at 121°C for 20 min at 15 psi (1.05 kg/cm2).
- Allow the medium to cool before adding antibiotics (optional).
Alternatively, commercially available 2x YT agar media formulations can be used. Weigh the required amount of the mixture and prepare the medium as instructed by the manufacturer.
Performance: The medium should exhibit luxurious growth of E. coli ATCC 23724 and ATCC 53868 strains when incubated between 35–37°C for 18–24 hours.
Storage
Sterilized 2x YT medium can be stored between 2–8°C. Do not freeze. When stored properly, it can last up to 6 months.
Points to be noted
Many supplements, like antibiotics, are heat-sensitive. Hence, they are not added to the medium before autoclaving. Instead, they are filter-sterilized and then added to the medium when it has cooled down sufficiently after autoclaving.