Magni:Protocols/Ligation
From OpenWetWare
< Magni:Protocols(Difference between revisions)
(→Ligation) |
Current revision (13:37, 2 February 2013) (view source) (→Ligation) |
||
| Line 11: | Line 11: | ||
*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
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.
