Haynes Lab:Notebook/Engineering PC-TFs/2014/05/21

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Setting up the ligation (CMV/MV9 & BL01)

  1. Calculate how many ng of insert you need to get a 4:1 ratio of insert molecules to 50 ng vector molecules
    X ng BL01 = (bp BL01 = 2520 / bp CMV/MV9 = 5197) x 4 x 50 ng CMV/MV9 = 96.979ng insert (BL01)
  2. Calculate how many μL of insert and vector you will need for each ligation:
    X μL BL01 = 96.979ng BL01 ÷ (BL01 concentration = 10.694ng/μL) = 9.06854μL
    X μL vector = 50 ng CMV/MV9 ÷ (CMV/MV9 concentration = 59.108ng/μL) = 0.8459μL
  3. Set up your ligation reaction(s) in sterile 0.5mL tubes as shown here:

  Ligation Negative Control
Insert DNA (X ng) 9μL none
Vector DNA (50 ng) 0.85μL same
10x Biolabs T4 Ligase Buffer 2.0 μl same
Biolabs T4 Ligase 1.0 μl same
dH2O 7.15μL 16.15μL
  20.0 μL total    same
  • Mix the reaction(s) thoroughly by flicking the tube.
  • Incubate at room temperature overnight.