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From 2012.igem.org

Contents 1 Make Super Optimal Broth (SOB) medium 2 Make selection plates for Spc (5x), Spc-Kan (5x), Kan (5x), and Neo-Kan (5x) 3 Make selection plates for Kan (20x) 4 Plate strains for O/N cultures from glycerol stocks 5 Make O/N cultures of ADP1 for natural competence

Make Super Optimal Broth (SOB) medium

• Add 20 (20.0) g Bacto-tryptone, 5 (5.0) g yeast extract, 0.584 (0.5849) g NaCl for 10 mM, and 0.186 (0.1864) g KCl for 2.5 mM
• Bring to 900 mL with dH₂O and mix with heating to dissolve the solids
• Add 1 mL 1 M NaOH to bring pH to ~7.0
• Autoclave
• Store at 4^o C once cooled to RT

Make selection plates for Spc (5x), Spc-Kan (5x), Kan (5x), and Neo-Kan (5x)

Note: the concentration of Kan used in these plates differs from the 12.5 μg/mL plates prepared previously by Spencer

• Prepare LB broth (pH 7.0) as usual in a 1-L E. flask, and pour 250 mL into two 500-mL flasks
• Autoclave
• Prepare each set of plates as follows:
  • LB - as usual (20x)
  • LB-Spc - add 10 μL/mL (2.5 mL for 10 plates) of 10 mg Spc/mL to get a concentration of 100 μg/mL; use a pipet to add 25 mL to each plate, set five aside (5x)
  • LB-Spc-Kan - add 0.2 μL/mL (5 μL total per plate) of 50 mg Kan/mL to get a concentration of 10 μg/mL (5x)
  • LB-Kan - add 0.2 μL/mL (50 μL for 10 plates) of 50 mg Kan/mL to get a concentration of 10 μg/mL; use a pipet to add 25 mL to each plate, set five aside (5x)
  • LB-Neo-Kan - add 2 μL/mL (50 μL total per plate) of 10 mg Neo/mL to get a concentration of 20 μg/mL (5x)
• Once hardened, store plates at 4^o C

Make selection plates for Kan (20x)

• Prepare LB broth (500 mL) as usual
• Autoclave
• Add 0.2 μL/mL (100 μL for 20 plates) of 50 mg Kan/mL to get a concentration of 10 μg/mL
• Add ~25 mL to each plate
• Once hardened, store plates at 4^o C

Plate strains for O/N cultures from glycerol stocks

• Plate Ac ADP1 WT, EcNR2, Bs 168, Bs PERM739, Bs PY79, and Bs 1A833 on their appropriate selection plates
• Incubate plates at 34^o C

Make O/N cultures of ADP1 for natural competence

Note: the transformation protocol calls for an O/N prepared from multiple colonies of a plate grown overnight, but a glycerol stock was used instead

• Prepare two O/N cultures of 2 mL each: one with LB medium, and the other with SOB medium, which has been shown in Ec at least to increase transformation efficiency at least two-fold.