User:Eric M. Walters/Notebook/Spring 2012/2012/04/11

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Enrichment of the real construct

  • Gel extracted the ~5.5kb band out of a gel run of the whole 50 μL ligation
    • Final conc: 6 ng/μL - eek!

PCR'd with the slightly interior v2 UpF & DnR primers to get more construct

4x50 μL

  • 1 μL acsA-up-F-2
  • 1 μL acsA-down-R-2
  • 1 μL Gel purified ligation product
  • 1 μL dNTPs
  • 1 μL Phusion
  • 10 μL 5x Buffer HF
  • 35 μL H2O

1:30 extension time, 72°C annealing temp. Run overnight.