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.invitrogen.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:
|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.
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.