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Team:Amsterdam/Notebook/Protocols/Ligations
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2. Incubate for 20min at 80C to heat kill.<br> | 2. Incubate for 20min at 80C to heat kill.<br> | ||
3. Use 2ul of ligation to transform into competent cells.<br> | 3. Use 2ul of ligation to transform into competent cells.<br> | ||
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Revision as of 19:42, 16 September 2011
Ligation
After following our digestion protocol a ligation can be performed.
Different ratios of vector:insert are used, to get the best result out of the ligation.
To check for self-closing vectors a vector-only sample will be ligated.
Materials
- PCR tubes/PCR plate
- vector and insert DNA
- dH20
- T4 DNA ligase
- T4 DNA ligase buffer
Note: All materials should be held on ice during preparation.
Protocol
| ratio 1:1 | ul |
|---|---|
| Vector | 1 |
| Insert | 1 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 6 |
| Total | 10 ul |
| ratio 1:2 | ul |
|---|---|
| Vector | 1 |
| Insert | 2 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 5 |
| Total | 10 ul |
| ratio 1:5 | ul |
|---|---|
| Vector | 1 |
| Insert | 5 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 2 |
| Total | 10 ul |
| Vector-only | ul |
|---|---|
| Vector | 1 |
| Insert | 0 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 7 |
| Total | 10 ul |
1. Incubate for ON at 16C.
2. Incubate for 20min at 80C to heat kill.
3. Use 2ul of ligation to transform into competent cells.
