Magni:Protocols/Ligation: Difference between revisions
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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. | ||
*1 ul of Ligase Buffer (Roche) | *1 ul of Ligase Buffer (Roche) | ||
*1 ul of T4 Ligase (Roche) | *1 ul of T4 Ligase (Roche) | ||
Mix well and incubate in the thermocycler (''T4Lig'' program) 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). | Heat-inactivate the enzyme at 65degC for 10 min in the thermocycler (''pretrasf'' program, reaction volume of 10 ul) before proceeding with transformation. | ||
Latest revision as of 10:37, 2 February 2013
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.
