TOPO Cloning
From Bridges Lab Protocols
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- PCR desired insert using Pfu polymerase. Add 1 uL of Taq and mix. Incubate 10min at 72C to add A to 3’ ends. Use immediately
- If PCR product is a single band, no need to purify further. If several bands, gel purify.
- Combine 4 uL PCR product, 1 uL Salt Solution, and 1 uL TOPO vector. Mix gently
- Incubate at room temperature for 5 min, place on ice.
- Thaw tube of OneShot cells on ice
- Add 2 uL reaction and incubate on ice 5-30 min
- Heat shock 30s at 42C without shaking
- Place on ice for 2 min
- Add 250 uL of room temperature SOC media
- Incubate at 37C for 1h
- Plate 50 uL on plate and grow overnight
