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Team:Amsterdam/Notebook/Protocols/Ligations
From 2011.igem.org
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(→Ligation) |
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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. | ||
| - | + | <br><br> | |
==Materials== | ==Materials== | ||
*PCR tubes/PCR plate | *PCR tubes/PCR plate | ||
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*T4 DNA ligase | *T4 DNA ligase | ||
*T4 DNA ligase buffer | *T4 DNA ligase buffer | ||
| - | < | + | <i>Note: All materials should be held on ice during preparation.</i> |
| - | + | <br><br> | |
| - | + | ||
==Protocol== | ==Protocol== | ||
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|}<br> | |}<br> | ||
| - | 1. Incubate | + | 1. Incubate ON at 16°C.<br> |
| - | 2. Incubate for 20 min at | + | 2. Incubate for 20 min at 80°C to heat kill.<br> |
| - | 3. Use | + | 3. Use 2 µl of ligation to transform into competent cells.<br> |
Revision as of 10:17, 19 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 ligation to transform into competent cells.
