Quint Lab:ligation (sticky ends)

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ligation

ligation mix:

  • x µl digested vector
  • y µl PCR fragment or digested insert
  • 1 µl l0x ligase buffer
  • 1 µl T4 DNA ligase [3 Weiss U/µl]
  • up to 10 µl dH2O

incubate O/N at 16ºC.

in most cases, a 1:1 or a 1:3 ratio work best


formular: [(ng vector) x (kb size of insert)] / (kb size of vector) x [molar ratio of (insert/vector)] = ng insert

example: 500bp insert to be ligated with 100ng of 3.0kb vector in a 3:1 ratio
→ [(100ng vector) x (0.5 kb of insert)] / [3.0 kb vector x (3/1)] = 50 ng insert

or simply use the vector:insert ratio calculator