From 2007.igem.org

• (CinR+HSL D. ES #4) + D-term
  • insert (CinR+HSL): 0.02 ug/uL = 20 ng/uL, 1530 bp
  • vector (D-term): 0.06 ug/uL = 60 ng/uL, 3269 bp
    • recommended vector mass: 100 ng to 150 ng
  • MI/MV = C * SI/SV
    • MI/MV = C * 1530/3269 =~ C/2
    • assume VI = X, VV = Y, (X*20)/(Y*60) = C/2, X/Y = 3C/2
    • total reaction volume is 20 uL and buffer is 10X
    • for each reaction we need
      • 1X buffer, thus volume of buffer is 20(uL)/10 = 2 (uL)
      • 1 uL T4 DNA ligase ([http://www.neb.com/nebecomm/products/productM0202.asp NEB, M0202])
      • X + Y = 20 - (1+2) = 17 = (1 + 1.5C)Y
      • choose C = 4, 7Y = 17, Y = 2.43, X = 6Y = 14.58
      • approximately Y = 3, X = 14, C = 3.3