Ligation Reaction

  • MQ water
  • 1.5 ml tubes
  • 10x T4 Ligation buffer
  • T4 Ligase
  • Insert
  • Vector
Reaction mixture:
Component 20 µl Final concentration
MilliQ water up to 20 µl
10 ligation buffer 2 µl 1x
Vector x µl 20-100 ng
Insert x µl 3:1 molar ratio over vector*
T4 DNA ligase 5 U/µl 2 µl 0.5 U/µl

* When restricted PCR products are ligated together, a 1:1 molar ratio is used.
All the reagents are added following the order listed in the table above [1].
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.