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Week 8: July 3-10, 2011

• Designed and ordered primers with BioBrick prefix and suffix overhangs to PCR amplify following constructs
  • RiAFP-His
  • eGFP-TEV-RiAFP-His
  • eGFP-TmAFP-His
  • eGFP-TEV-TmAFP-His
  • TmAFP-His
• Set up PCR amplification with Phusion polymerase of aforementioned constructs, verified successful amplification: (show figure)
• Digest PCR fragments using XbaI and PstI, digest BioBrick BBa_I712074 (contains T7 polymerase) with SpeI and PstI
• Ligate using T4 ligase, transform cells and plate on Amp plates (backbone of plasmid is AmpR and KanR)
• Verification of successful ligation using colony PCR