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From 2011.igem.org

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Notebook

Run through for abstract and scheduling:

Start with plasmid with taq gene and pst1 gene

Remove pst1 site from taq coding sequence

• In order to remove the restriction site, do site directed mutagenesis (1day)
• PCR
• Digestion
• Transformation
• Screening (1day)
  • Dilute DNA
  • Colony PCR individually
  • Digestion of PCR product with pst1
  • Run gel ~2500bp

Add biobrick prefix/suffix to taq coding sequence

• PCR and add the prefix and suffix as primers (one prefix has an extra AG-make sure to use the correct one) (1-2 days)
  • Run gel
  • Cut out of gel
• Cut with restriction enzyme
• Clean up DNA
• Ligation with vector (could be vector with the terminator sequence, promoter and RBS)
• Transformation

Add a promoter, transcriptional terminator, ribosome binding site (RBS)

• Screen colonies (1 day)
  • Colony PCR
  • Restriction Digestion
  • Clean up DNA
• Sequence (1 day)

Make taq protein

Compare it to other enzymes and make sure it works