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Description
Instructions are provided to TOPO Clone your PCR product into pCR-Blunt II-TOPO and transform the reaction into chemically competent E. coli cells. For transformation of electrocompetent E. coli cells, a diagram of the multiple cloning site, and detailed instructions, refer to the Zero Blunt TOPO PCR Cloning Kit manual available from www.thermofisher.com or Technical Support.
Producing Blunt PCR Products
Produce blunt-end PCR products using a thermostable proofreading polymerase and your own protocol. End the PCR reaction with a final 7 to 30 minutes extension step.
TOPO Cloning Reaction
- Set up the following 6 μL TOPO Cloning reaction:
| Reagent | Amount* |
| Fresh PCR Product | 0.5 to 4 μL |
| Salt Solution | 1 μL |
| Sterile Water | add to a total volume of 5 μL |
| pCR-Blunt II-TOPO | 1 μL |
| Final Volume | 6 μL |
* For transformation of chemically competent E. coli only.
- Mix gently and incubate for 5 minutes at room temperature.
- Place tubes on ice. Proceed to Transformation and Analysis.
Protocols to transform chemically competent cells and to analyze positive clones are provided below. If you wish to transform electrocompetent cells, refer to the Zero Blunt TOPO PCR Cloning Kit manual for instructions.
One Shot Chemical Transformation
- Thaw on ice 1 vial of One Shot E. coli cells for each transformation.
- Add 2 μL of the TOPO Cloning reaction to a vial of One Shot E. coli and mix gently.
- Incubate on ice for 5–30 minutes.
- Heat-shock the cells for 30 seconds at 42°C without shaking.
- Add 250 μL of room temperature S.O.C. medium to the cells.
- Cap the tubes and shake at 37°C for 1 hour.
- Spread 10–50 μL from each transformation on prewarmed LB plates containing 50 μg/ml kanamycin or prewarmed Low Salt LB plates containing 25 μg/ml Zeocin. Refer to the Zero Blunt TOPO PCR Cloning Kit manual for a recipe for Low Salt LB medium.
- Incubate plates overnight at 37°C.
- An efficient TOPO Cloning reaction should produce several hundred colonies. Pick ~10 colonies for analysis. Proceed to Analyzing Positive Clones.
Analyzing Positive Clones
- Culture the 10 colonies overnight in LB medium containing 50 μg/mL kanamycin or Low Salt LB medium containing 25 μg/mL Zeocin.
- Isolate plasmid DNA using your method of choice. For ultrapure plasmid DNA, we recommend the PureLink HQ Mini Plasmid Purification Kit (Catalog no. K2100-01).
- Analyze the plasmid by restriction analysis.