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Set up the ligation mix according:

Reagents 1:3(μl) 1:5(μl) Vector(μl)
Vector 1 1 1
Insert 3 5 N/A
10X BUFFER 1 1 1
T4 Ligase 1 1 1
H2O 4 2 7
Total Volume 10.0 10.0 10.0

For sticky end ligation, incubate the tube containing above mentioned chemicals on the bench for a period of 3-4 hours or overnight.

Important point to note is that 1:3 dilution means that during ligation, for 1 unit of vector, there are 3 units of insert and in 1:5 ligation, for 1 unit of vector there is 5 units of insert.


  • For standard ligation, 100-200 ng of vector DNA is required and this can be checked using nanodrop protocol (refer to Nanodrop Spectrophotometer protocol).
  • Ligation calculation can be used to calculate the amount of vector and insert required for the ligation but 1:3 or 1:5 ligation ratios are used more frequently.

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