Knight:TempliPhi

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Current revision (15:43, 18 November 2007) (view source)
 
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#Heat to 65°C for 10 mins.
#Heat to 65°C for 10 mins.
#Cool to 4 °C.
#Cool to 4 °C.
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#Use 1&mu;L to submit for [[Knight:Sequencing DNA|sequencing]]. <-- ''This is what we used previously when sequencing on the Knight lab sequencer.''
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#Send DNA for [[Knight:Sequencing DNA|sequencing]].  
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#* Use 2&mu;L in 12&mu;L for sequencing at the biopolymers facility
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#* Previously, for sequencing on the Knight lab sequencer, we used 1&mu;L
[[Category:Protocol]]  
[[Category:Protocol]]  
[[Category:In vitro]]
[[Category:In vitro]]
[[Category:DNA]]
[[Category:DNA]]

Current revision

< TempliPhi

Materials

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
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