Magni:Protocols/Ligation

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Ligation

After DNA quantification with Nano-Drop, add to a 0.2-ml PCR tube (10 ul final volume):

• 20-40 ng of vector DNA
• ng of insert: 6 (insert length/vector length) (ng of vector DNA)
  • NOTE: the factor 6 can be decreased up to a factor 2
• Milli-Q water up to 8 ul.
• 1 ul of Ligase Buffer (Roche)
• 1 ul of T4 Ligase (Roche)

Mix very well and incubate in the thermocycler (T4Lig program, reaction volume of 10 ul) at 16degC overnight.

Heat-inactivate the enzyme at 65degC for 10 min in the thermocycler (pretrasf program, reaction volume of 10 ul) before proceeding with transformation.