User:Eric M. Walters/Notebook/Spring 2012/2012/05/21

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Producing the barcoded KO assembly

  • I'll amplify the acsA-up and acsA-down regions with the half barcode sequence, then digest, and ligate together

Amplifications

  • Primer annealing is 68°C (NEB calc)
    • These are a 1:10 dilution of the stocks
  • 500bp amplification, I'll give it :15 seconds extension
  • 10 uL HF buffer
  • 1 uL dNTP
  • 1 uL Phusion
  • 1 uL fwd primer
    • acsA-dnF-BCback-SphI or acsA-upF2
  • 1 uL rev primer
    • acsA-dnR2 or acsA-UpR-BCfront-SphI
  • 1 uL 7002 gDNA
  • 35 uL H20