SEED/2010/Day 6

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Contents

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
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