User:Eric M. Walters/Notebook/Spring 2012/2012/03/13

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acsA-up-down, accBCDA digest

Concentrations:

  • acsA-up: 119 ng/μL
  • acsA-down: 154 ng/μL
  • accBCDA: 211 ng/μL

Volumes to digest:

  • acsA-up: 31 μL (all of it)
  • acsA-down: 30 μL
  • accBCDA: 19 μL

Volumes of water (& 5 μL buffer 4):

  • acsA-up: 13 μL
  • acsA-down: 14 μL
  • accBCDA: 24 μL

Enzymes:

  • acsA-up: 1 μL EcoRI-HF
  • acsA-down: 1 μL BamHI-HF
  • accBCDA: 1 μL EcoRI-HF, 1 μL BamHI-HF

Run in the iGEM lab's 37 waterbath from 3:30-6:00, gel extracted.

Ligation (50 μL)=

Concentrations (up, acc, down): 48, 23, 63 ng/μL So reaction was:

  • 3.5 up
  • 21 accBCDA
  • 3 down
  • 5 buffer
  • 5 ligase

16°C O/N