Knight:TempliPhi

< TempliPhi

Materials

• illustra TempliPhi™ 100/500 Amplification Kit
• 0.6mL PCR tubes

Procedure

1. Thaw sample buffer (red cap) and reaction buffer (blue cap) on ice.
2. Transfer 5 μL of sample buffer to small PCR tube for each template to be amplified.
3. 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)
4. Heat sample to 95°C for 3 mins to denature and cool to 4°C.
5. Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions.)
6. Transfer 5 μL TempliPhi premix to cooled sample.
7. 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.
8. Heat to 65°C for 10 mins.
9. Cool to 4 °C.
10. 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