Team:Osaka/Protocols

Protocols

• Preparation of competent cells for heat-shock transformation
  • Preparation

1. SOB medium 1L(-> final conc.)
   • Bacto Tryptone 20g(-> 2.0%)
   • Bacto Yeast Extract 5g(-> 0.5%)
   • 5M NaCl 2ml(-> 10mM)
   • 2M KCl 1.25ml(-> 2.5mM)
   • Add miliQ water 990ml to all miture and sterilize by autoclave. Before using, add sterilized Mg2 solution(1M MgSO4, MgCl2) 10 ml.
2. SOC medium
   • Add sterilized 2M glucose 1 / 100 volume to SOB medium, and sterilize by filtration using 0.22 um microfilter. Store at 4℃.
3. Tranformation buffer, TB 1L(-> final conc.)
   • PIPES 3.0g(-> 10mM)
   • CaCl2 2H2O 2.2g(-> 15mM)
   • KCl 18.6g(-> 250mM)

• Transformation by heat-shock
• Plasmid DNA miniprep
• 3A BioBrick assembly
• PCR cloning
• PCR site-directed mutagenesis
• Extraction of genome DNA for PCR cloning