Terrific Broth (TB)

Terrific Broth (TB)

• 12 g/L tryptone (1.2% m/V)
• 24 g/L yeast extract (2.4% m/V)
• 5.04 g/L glycerol (4 mL/L, 4.34 mM, 0.4% V/V)
• 17 mM KH₂PO₄, 2.31 g/L
• 72 mM K₂HPO₄, 12.54 g/L
• (opt.) 15 g/L agar (1.5% m/V)
• (opt., nontrad.) 5–10 mM 1 M MgSO ₄  or MgCl ₂
  

It was originally suggested to separately autoclave phosphate buffer and agar. It was recently found that phosphate and agar react to form H₂O₂ , but  E. coli are less, if at all, significantly impacted due to high catalase expression. Commercial mixes do not separate the two, yet instruct autoclaving. There is also reason for separately autoclaving the glycerol, as glycerol partakes in Maillard reactions with tryptone/yeast extract amines, though probably less so than sugars do due to its lack of carbonyls. Maillard reactions sequester nutrients and form products that decompose into growth-inhibitory compounds. Sterile 50% glycerol can be added after autoclaving.

Magnesium is deficient in LB. It has been found that in peptone and tryptone broths, Mg²⁺ is necessary for robust growth, especially when glucose is provided. There is a substantial growth improvement with 1 mM MgCl₂ even in phosphate-buffered tryptone broth, proving the insufficiency of contaminant Mg²⁺ in phosphate salts, though phosphate does bring in substantial contaminant trace minerals. 1–10 mM MgSO₄ or MgCl₂ can be supplemented to any peptide broth for best growth and minimal cell stress.

2×YT Medium

• 16 g/L tryptone (1.6% m/V)
• 10 g/L yeast extract (1.0% m/V)
• 5 g/L NaCl (0.5% m/V)
• (opt.) 15 g/L agar (1.5% m/V)
• (opt., nontrad.) 5–10 mM 1 M MgSO₄ or MgCl₂

Recovery Medium:

Shyam rationalizes that TB or 2×YT supplemented with 0.4% V/V glucose and MgCl₂ and/or MgSO₄ to a final 10–20 mM would be just as good as SOC for transformation recovery. SOB/SOC, has 10 mM MgCl₂ and 10 mM MgSO₄ to help rebuild the cation-associated E. coli outer membrane LPS, and glucose is an ideal carbon source.

Procedure for 1 L TB

1. Dissolve and autoclave on liquid cycle, 15–20 min, 15 psi:
   1. 12 g tryptone
   2. 24 g yeast extract
   3. 4 mL glycerol (or 8 mL 50%_V/V glycerol)
      (or add after autoclaving) 
   4. (opt.) 15 g agar
   5. (sug.) 5 mL 2 M MgCl₂ or MgSO₄
   6. final 900 mL diH₂O
2. Prepare or obtain 100 mL 10× TB buffer (0.17 M KH₂PO₄ 0.72 M K₂HPO₄)
   Dissolve and autoclave on liquid cycle, 15 min, 15 psi:
   1. 2.31 g KH₂PO₄
   2. 12.54 g K₂HPO₄
   3. ≈90 mL diH₂O, adjusted to 100 mL final
3. When 900 mL nutrient solution has cooled ≤60°C, add 100 mL sterile buffer.