SEED/2009/Day 6

Morning

• set up 5ul ligations with plasmid, each of 6 promoters, plus one RBS.reporter
  • 0.5ul T4 ligase buffer
  • 10ng plasmid
  • 10ng RBS.reporter
  • 0.2ul promoter
  • 0.25ul T4 ligase
• Incubate at room temperatures over lunch
• pour plates (LB + amp + IPTG + X-gal)

Afternoon

• Do quiz on units
• Individual meetings with instructor to discuss final projects
• transformation of 6 ligations + 10pg pUC19 into chemically competent E. coli Top10 cells
  • use 100ul of cells and 2ul of the ligation mix
  • Leave on ice for 30 minutes
  • Heat shock for 45 sec @ 42C
  • Place back on ice
  • Add 300ul SOC
  • Incubate @ 37C for 30 min
  • plate entire transformation mix
• Incubate at 37C

Instructor Preparation

• oligos with different promoters from this set registry:Part:BBa_J23101 pre-cut with EcoRI/SpeI
  • T4 PNK top/bottom oligos
    • 10ul: 1ul T4 ligase buffer, 1ul 50uM oligo, 8ul water, 0.25ul T4 PNK
    • 4h@37, 10m@65
  • mix and anneal pairs

Instructor Post-prep

• Store plates in fridge