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Revision as of 02:13, 28 October 2010

• Home
• Project
  • About The Toolkit
  • Oursystem
  • Producing The Antigen
  • Future Applications
  • Safety
• Toolkit
• Why Pichia
  • Pichia Vs Saccharomyces
  • Easy and Inexpensive to Culture
  • High production of foreign Protein while low levels of endogenous protein
  • Pichia pastoris versus Esherichia coli
• Protocols
  • Preparation Of Electrocompetent Cells
  • Preparation of Heat Shock Competent Cells
  • Transformation Using Electroporation
  • Transformation Using Heat Shock
• The Team
• Notebook

How we made our parts

Part Design: Using primers that contain Biobrick ends, parts were isolated from Pichia pastoris chromosomal DNA with PCR reactions. To increase the likelihood of gaining a successful product, a systematic approach was applied for primer design. Two sets of primers were constructed for each part using IDT®’s PrimerQuest and Oligoanalyzer programs. The first set added an Xpa I site on the forward and a Spe I site on the reverse. The second set added EcoRI, Not I and Xba I sites on the forward and Spe I, Not I and Pst I sites on the reverse. In this way, hybrid primer combinations were assembled providing a total of four PCR products. With this system, we have successfully acquired a biobrick prospective product for each part.