User:Eric M. Walters/Notebook/Spring 2012/2012/03/14

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Purifying ligation

Extracted ~5.5kb band (maybe 6ish) Nanodropped to get a horrible concentration (5.5 ng/μL) but >2 260/280? Visible at (and only at) correct size on gel, used as template for below PCR reaction

Amplifying insert

2x50 μL reactions at each 57 (taq) and 72 (phusion) annealing temps, based on NEB's Tm calc

  • 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