From 2007.igem.org

size (bp) of insert part (SI)
size (bp) of vector part (SV)
concentration (ug/uL) of insert (CI)
concentration (ug/uL) of vector (CV)
volume (uL) of insert for gel separation (VI)
volume (uL) of insert for ligation (L)
ligation ratio

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• total digestion volume is 20 uL (typical)
  • enzyme 1 and enzyme 2 (1uL * 2)
  • 10X buffer (2uL)
  • BSA (2uL)
  • plasmid + ddH2O (14uL = VI + water, 1 <= VI <= 14)
  • for vector
    • we assume 300 ng for digestion and 90% loss
    • thus, amplify 10X from 300 ng to 3,000 ng = 3 mg
    • 3,000 ng = CV (ug/uL) * 1000 (ng/ug) * DVV, DVV (digested vector volume) = 3/CV

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• total gel separation volume is 50 uL

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• total ligation volume is 10uL
  • (MI/MV) = R * (SI/SV)
  • assume mass of vector (MV) is 300 ng
    • MI = R * (SI/SV) * MV = 300R * (SI/SV) (ng)
  • 10X buffer (1uL)
  • ligase (1uL)
  • VV + L + water (8uL)

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• formula
  • 300 * SI * 3 * 10 (損失因子) / SV = CI * VI * L /50
  • vector weight is 300 ng
  • SI (bp) is size of insert
  • SV (bp) is size of vector
  • VI (uL) is volume of insert
  • VV (uL) is volume of vector
  • CI (ng/uL) is concentration of insert
  • CV (ng/uL) is concentration of vector
  • R is ranged from 1 to 10