- Thaw sample buffer (red cap) and reaction buffer (blue cap) on ice.
- Transfer 5 μL of sample buffer to small PCR tube for each template to be amplified.
- Add template to sample buffer
- Dilute 1μL saturated overnight culture in 10-100 μL water. Use 0.2-0.5 μL.
- Use small portion of a colony. Avoid transferring any agar.
- Dilute 1μL glycerol stock in 50μL water. Use 0.2-0.5 μL.
- Use 1pg-10ng of purified plasmid DNA (volume < 0.5μL)
- Heat sample to 95°C for 3 mins to denature and cool to 4°C.
- Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions.)
- Transfer 5 μL TempliPhi premix to cooled sample.
- Incubate at 30°C for 4-18 hours.
- Incubate overnight for optimal results but 4 hours should be sufficient if there is not too much inhibitory material like agar or rich medium.
- Heat to 65°C for 10 mins.
- Cool to 4 °C.
- Send DNA for sequencing.
- Use 2μL in 12μL for sequencing at the biopolymers facility
- Previously, for sequencing on the Knight lab sequencer, we used 1μL