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From 2012.igem.org

Contents 1 Prepare plates of Bs strains to make competent 2 Prepare plates of template strains of Ec 3 Prepare O/N cultures from Pedraza-Reyes strains for glycerol stocks 4 Transform pBR322 into Ec XL1-Blue competent cells

Prepare plates of Bs strains to make competent

• As per the two-step transformation procedure in the "Protocols" page, plate the following strains as lawns:
  • Bs1A833 (ΔmutSL::Spc) from glycerol stock
  • Bs 168 (auxotroph for Trp) from glycerol stock
  • Bs PY79 (WT) from glycerol stock
  • Bs PERM739 (ΔmutS::Neo)
• Incubate at 34^o C overnight

Prepare plates of template strains of Ec

• Plate the following Ec strains for individual colonies to use as template DNA for construct assembly:
  • EcNR2 (lacZ)
  • EcFI5 (cat*)

Prepare O/N cultures from Pedraza-Reyes strains for glycerol stocks

• Inoculate PERM311 (168), PERM704 (ΔyfhQ::Spc), and PERM739 (ΔmutS::Neo) from respective selection plates into 2 mL LB broth with appropriate antibiotics
• Incubate at 34^o C overnight

Transform pBR322 into Ec XL1-Blue competent cells

• Thaw two 200-μL aliquots of XL1-Blue competent cells on ice
  • To one of the tubes, add 2 μL 1:10 diluted pBR322 (obtained from Natalie Ma) and stir gently with pipette tip
  • To the other tube, add 2 μL sterile dH₂O and stir gently with pipette tip
• Incubate both tubes on ice for 30 minutes
• Heat shock tubes for 45 seconds in 42^o C water bath
• Incubate on ice for 2 minutes
• Add 500 mL LB broth to each tube and incubate for an hour at 34^o C with shaking
• Spread 100 μL of culture onto LB-Amp selection plates with sterile glass spreader and incubate overnight at 34^o C