SEED/2010/Day 3

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  • each group will miniprep 1.5ml of pSB4A5.I52001 and pSB1A3.E0050 cultures
    • elute in 30ul


  • spec miniprep concentrations
  • Set up one 50ul digest of pSB4A5 with EcoRI and PstI
    • 5ul NEB2
    • 0.5ul BSA
    • 2.5ug pSB4A5 miniprep
    • 1ul EcoRI
    • 1ul PstI
  • leave in 37C incubator overnight


  • Restriction digest and gel electrophoresis


  • What are you going to learn and do in this class?
    • Cloning Process Overview
    • Characterization

Instructor Preparation

  • grow up cultures of pSB4A5.I52001 (destination vector) and 1A3.E0050 (PCR template)
  • prepare minipreps in case student prep concentrations are too low

Instructor Post-prep

  • heat kill and store miniprep digests in freezer