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Team:Tuebingen/NotebookProtocols
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=== Chemo-competent cells === | === Chemo-competent cells === | ||
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Ligation for TA-cloning of PCR products | Ligation for TA-cloning of PCR products | ||
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Mix all reagents in a 0.5ml tube. Incubate reaction at 4°C over night. | Mix all reagents in a 0.5ml tube. Incubate reaction at 4°C over night. | ||
| - | + | === Ligation === | |
Ligation for digested parts and vectors. | Ligation for digested parts and vectors. | ||
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Revision as of 14:31, 20 September 2012
Contents |
Protocols
Chemo-competent cells
pGEM Ligation
Ligation for TA-cloning of PCR products
| Component | Volume |
|---|---|
| 2X Rapid Ligation Buffer | 5µl |
| pGEM vector | 0.5µl (25ng) |
| PCR product | 3.5µl |
| T4 DNA ligase | 1µl (3 Weiss units) |
Mix all reagents in a 0.5ml tube. Incubate reaction at 4°C over night.
Ligation
Ligation for digested parts and vectors.
| Component | Volume |
|---|---|
| 10X T4 DNA Ligase Buffer | 1µl |
| vector DNA | 1µl (20-100ng) |
| insert DNA | 5µl (up to 5:1 molar ratio insert to vector) |
| T4 DNA ligase | 1µl (1 unit) |
| water | 2.5µl |
Mix all reagents and incubate at 22°C for 1h.
