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Team:Amsterdam/Notebook/Protocols/Ligations
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| - | After following our digestion protocol a ligation can be performed.<br> | + | After following our [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Digestion digestion protocol] a ligation can be performed.<br> |
Different ratios of vector:insert are used, to get the best result out of the ligation.<br> | Different ratios of vector:insert are used, to get the best result out of the ligation.<br> | ||
To check for self-closing vectors a vector-only sample will be ligated. | To check for self-closing vectors a vector-only sample will be ligated. | ||
| Line 110: | Line 110: | ||
1. Incubate ON at 16°C.<br> | 1. Incubate ON at 16°C.<br> | ||
2. Incubate for 20 min at 80°C to heat kill.<br> | 2. Incubate for 20 min at 80°C to heat kill.<br> | ||
| - | 3. Use 2 µl of ligationmix to transform into competent cells.<br> | + | 3. Use 2 µl of ligationmix to transform into [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Transforming_Competent_Cells chemically competent cells] or [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Transforming_electrocompetent_Cells electro competent cells].<br> |
{{:Team:Amsterdam/Footer}} | {{:Team:Amsterdam/Footer}} | ||
Latest revision as of 00:36, 22 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 | µl |
|---|---|
| Vector | 1 |
| Insert | 1 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 6 |
| Total | 10 µl |
| ratio 1:2 | µl |
|---|---|
| Vector | 1 |
| Insert | 2 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 5 |
| Total | 10 µl |
| ratio 1:5 | µl |
|---|---|
| Vector | 1 |
| Insert | 5 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 2 |
| Total | 10 µl |
| Vector-only | µl |
|---|---|
| Vector | 1 |
| Insert | 0 |
| T4 DNA ligase buffer | 1 |
| Ligase | 1 |
| H2O | 7 |
| Total | 10 µl |
1. Incubate ON at 16°C.
2. Incubate for 20 min at 80°C to heat kill.
3. Use 2 µl of ligationmix to transform into chemically competent cells or electro competent cells.
