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)
   • After this mixture is suspended in about 950ml sterilized water, control　the pH to 6.7-6.8 by 5N KOH (5M KOH). In low pH, this mixture cannot dissolve. Next, add MnCl2 4H2O (10.9g) to the solution, so as to be final concentration 55mM. Ajust it to 1L and sterilize by filtration using 0.22 um microfilter. Store at 4℃.
4. Liquid nitrogen
5. DMSO (dimethyl sulfoxide)

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