Magni:Protocols/Ligation

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(Ligation)
Current revision (13:37, 2 February 2013) (view source)
(Ligation)
 
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*1 ul of T4 Ligase (Roche)
*1 ul of T4 Ligase (Roche)
-
Mix well and incubate in the thermocycler (''T4Lig'' program, reaction volume of 10 ul) at 16degC overnight.
+
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.
Heat-inactivate the enzyme at 65degC for 10 min in the thermocycler (''pretrasf'' program, reaction volume of 10 ul) before proceeding with transformation.

Current revision

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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.
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