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Team:Tuebingen/NotebookProtocols
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Protocols
Chemo-competent cells
pGEM Ligation
Ligation for TA-cloning of PCR products
| Component | Volume |
|---|---|
| 2X Rapid Ligation Buffer | 5 µl |
| pGEM vector | 0.5 µl (25ng) |
| PCR product | 3.5 µl |
| T4 DNA ligase | 1 µl (3 Weiss units) |
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
Ligation
Ligation for digested parts and vectors
| Component | Volume |
|---|---|
| 10X T4 DNA Ligase Buffer | 1 µl |
| vector DNA | 1 µl (20-100 ng) |
| insert DNA | 5 µl (up to 5:1 molar ratio insert to vector) |
| T4 DNA ligase | 1 µl (1 unit) |
| water | 2.5 µl |
Mix all reagents and incubate at 22°C for 1 hour.
Chemotransformation
| Component | Volume |
|---|---|
| chemo-competent E. coli | 100 µl |
| plasmid DNA | up to 10 µl (max. 1/10 of volume) |
- Add plasmid DNA to cell culture.
- Incubate for 30 min on ice.
- Heat shock for 90 sec at 42°C.
- Add 900 µl LB.
- Let the bacteria grow at 37°C for at least 1 hour.
Restriction digest
control digest
preparative double digest
plasmid linearization
PCR
| Component | Volume |
|---|---|
| Taq/Pfu buffer | 5 µl |
| Taq/Pfu polymerase | 1 µl |
| primer forward | 0.5 µl (100 pmol/µl) |
| primer reverse | 0.5 µl (100 pmol/µl) |
| dNTPs | 2.5 µl (200 µM) |
| template DNA | 1 µl |
| water | 36 µl |
PCR conditions
| Step | Duration | Settings |
|---|---|---|
| 1 | 2 min | 94°C |
| 2 | 45 sec | 94°C |
| 3 | 30 sec | gradient or annealing temperature |
| 4 | 90 sec | 72°C |
| steps 2-4: 30 cycles | ||
| 5 | 7 min | 72°C |
| 6 | (hold) | 4°C |
Gel electrophoresis
TAE buffer 50x
| Component | Volume |
|---|---|
| 0.05 M EDTA | 18.61 g |
| 1 M acetic acid | 60.05 g |
| 2 M Tris | 242.28 g |
| water | 1 l |
Set to pH 8.5.
LB medium
incl. Agarplatten
