Magni:Protocols/Ligation

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(Ligation)
(Ligation)
Line 5: Line 5:
After DNA quantification with Nano-Drop, add to a 0.2-ml PCR tube (10 ul final volume):
After DNA quantification with Nano-Drop, add to a 0.2-ml PCR tube (10 ul final volume):
*20-40 ng of vector DNA
*20-40 ng of vector DNA
-
*6 (insert length/vector length) (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
**NOTE: the factor 6 can be decreased up to a factor 2
*Milli-Q water up to 8 ul.
*Milli-Q water up to 8 ul.
-
*Milli-Q water up to 20.5 ul.
 
*1 ul of Ligase Buffer (Roche)
*1 ul of Ligase Buffer (Roche)
*1 ul of T4 Ligase (Roche)
*1 ul of T4 Ligase (Roche)
Line 14: Line 13:
Mix well and incubate in the thermocycler (''T4Lig'' program) at 16degC overnight.
Mix well and incubate in the thermocycler (''T4Lig'' program) at 16degC overnight.
-
Heat-inactivate the enzyme at 65degC for 10 min in the thermocycler (''pretrasf'' program).
+
Heat-inactivate the enzyme at 65degC for 10 min in the thermocycler (''pretrasf'' program) before proceeding with transformation.

Revision as of 10:22, 14 September 2012

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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 well and incubate in the thermocycler (T4Lig program) at 16degC overnight.

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