Team:Amsterdam/Notebook/Protocols/Ligations

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Ligation

After following our digestion protocol a ligation can be performed.
Different ratios of vector:insert are used, to get the best result out of the ligation.
To check for self-closing vectors a vector-only sample will be ligated.

Materials

  • PCR tubes/PCR plate
  • vector and insert DNA
  • dH20
  • T4 DNA ligase
  • T4 DNA ligase buffer

Note: All materials should be held on ice during preparation.

Protocol

ratio 1:1 µl
Vector 1
Insert 1
T4 DNA ligase buffer 1
Ligase 1
H2O 6
Total 10 µl

ratio 1:2 µl
Vector 1
Insert 2
T4 DNA ligase buffer 1
Ligase 1
H2O 5
Total 10 µl

ratio 1:5 µl
Vector 1
Insert 5
T4 DNA ligase buffer 1
Ligase 1
H2O 2
Total 10 µl

Vector-only µl
Vector 1
Insert 0
T4 DNA ligase buffer 1
Ligase 1
H2O 7
Total 10 µl

1. Incubate ON at 16°C.
2. Incubate for 20 min at 80°C to heat kill.
3. Use 2 µl of ligationmix to transform into competent cells.