SEED/2008/Day 3

Morning

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

Afternoon

• 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

Quiz

• Restriction digest and gel electrophoresis

Lecture

• 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