Magni:Protocols/Ligation: Difference between revisions
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(New page: {{Magni}} <div style="padding: 10px; width: 720px; border: 5px solid #000000;"> = Ligation = After DNA quantification with Nano-Drop, add to a 0.2-ml PCR tube (10 ul final volume): *20-40...) |
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*20-40 ng of vector DNA | *20-40 ng of vector DNA | ||
*6 (insert length/vector length) (ng of vector DNA) | *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. | *Milli-Q water up to 8 ul. | ||
*Milli-Q water up to 20.5 ul. | *Milli-Q water up to 20.5 ul. | ||
Revision as of 07:18, 14 September 2012
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
- 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.
- Milli-Q water up to 20.5 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).
