LB (Lysogeny Broth/Agar)

1. Combine the following in an appropriate sized flask or bottle:

   1. For broth:

      • 25 g/L LB powder
        or from separate components:
        • 10 g/L tryptone
        • 5 g/L yeast extract
        • 10 g/L NaCl for LB-Miller (common).  5 g/L for LB-Lennox. 0.5 g/L for LB-Luria.

   2. For agar:

      • 40 g/L LB agar powder
        or from separate components:
      • • 10 g/L tryptone
        • 5 g/L yeast extract
        • 10 g/L NaCl for LB-Miller (common). 5 g/L for LB-Lennox. 0.5 g/L for LB-Luria.
        • 15 g/L agar

   3. For LB³⁰: Add an additional 25 g/L yeast extract to either recipe for a final 30 g/L.

   4. Enhancements:
      1. MgCl₂ (or MgSO₄): add 5–10 mM from an autoclaved 1 M stock. Improves growth yield and rate, reduces cell stress and protein acetylation.^{(2,4,5, Notes)}.
      2. Buffer: add 1× M9 salts from an autoclaved 5× or 10× stock. Reduces cell stress, purportedly.
      3. Carbohydrate: add 5 g/L (0.5% _{m /V} ) glucose or 10 g/L glycerol. Improves cell yield, reduces cell stress. Glycerol catabolism is less acidifying than glucose.
   5. Water: Milli-Q water or dionized water up to final volume.
2. Close and shake to partially combine, or stir with stir bar. Flasks can be closed with parafilm or gloved hand.
   For agar dishes (plates), adding a stir bar is advised for quicker and uniform cooling prior to pouring, unless using a 50–55° water bath.
   Remove stir bar from broth; it isn't needed later.
3. Sterilize by autoclaving for 15–20 min, 15 psi on the liquid cycle; 15 min for ≤1 L volume(s) and 20 min for >1 L volume(s).
4. Allow the medium to cool to 50–55°C. Add any antibiotics or other thermosensitive additives, e.g. inducers, only when the vessel's temperature has fallen to 50–55°C. Swirl thoroughly to distribute additives if not using a stir bar.
   • Cooling can be sped and made uniform by stirring with a stir bar added prior to autoclaving. Cooling rate can be maximized to ≈10 min for 1 L volumes by immersing the flask/bottle with stir bar in a small autoclave bin filled with water and set over a stir plate, stirring rapidly. Stir bar is essential, or else agar will congeal.
   • For agar, monitor temperature closely using a tape thermometer held against the vessel surface over the medium, or an infrared thermometer pointed at a dry vessel surface in contact with the medium.
   • Plates ought to be poured soon after medium reaches 50°C to allow time for pouring before agar congeals.
   • Immersing the vessel in a 50°C water bath, as made using a "sous vide" immersion circulator clipped to the side of an autoclave bin filled with deionized water, will not maximize the cooling rate of the medium but will allow the most time flexibility when dispensing agar, as the agar will cool to the proper temperature and remain so until one is ready to add additives and until one is done dispensing the agar.
   • Polystyrene Petri dishes may deform if agar is hotter than 70°C.
5. For agar, dispense ≈15–20 mL into each Petri dish.Work quickly to avoid agar congealing in the vessel.
   1. Pouring is fastest, but less accurate: stop pouring when ≈¾ dish is covered; then let the remainder fill, slightly swirling the dish or stack of dishes if needed.
   2. For more accuracy, use a 50 mL pipette to dispense, though slower and more prone to premature congealing and pipette clogging.
   3. Stacks of plates can be efficiently filled from the bottom up: fill a dish with one hand while holding its lid below the remainder empty stack above it in the other hand.
   4. Bubbles can be eliminated by very briefly (<1 s) passing a flame over the surface.

Cloning Broth: LB³⁰ _enh

Greatly enhances low- and high-copy plasmid yields  ⁽⁴⁾ . Even better with 10 mM Mg and (maybe) 1× M9 salts, Shyam's standard cloning broth.

LB³⁰ (regular) just has added yeast extract, compared to LB; LB ³⁰ _Mg has additional Mg; LB ³⁰ _enh has additional Mg and buffering. ^{(Shyam, 6)}.

• 878 mL/L Milli-Q water
  • Add half volume first. Rinse bottle sides with remaining half to dissolve stuck powder.
  • Using scale for water saves you from measuring volume.
• 25 g/L LB powder
  or from separate components:
  • 10 g/L tryptone
  • 5 g/L yeast extract
  • 10 g/L NaCl
• 25 g/L yeast extract (additionally, for final 30 g/L)
  
  Swirl to incorporate powders into half the water. Then add remaining water, washing down sides. Autoclave 15 min for ≤1 L volumes. After cool enough to handle, add:

• 10 mL/L 1 M MgCl₂ (autoclaved). Thoroughly swirl to mix; then add:
• 100 mL/L 10× M9 or TB Salts. Swirl to mix.
  • I have been using old 10× M9 salts otherwise to be wasted. Has not been tested if buffering improves "neutral" plasmid yields or just more toxic plasmid yields.
• Only when needed, 0.4–1% glucose or glycerol can be added for improving growth of strains with toxic constructs. Buffering must be used with simple carbon sources.