Knight:TempliPhi

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#*Use small portion of a colony.  Avoid transferring any agar.
#*Use small portion of a colony.  Avoid transferring any agar.
#*Dilute 1μL glycerol stock in 50μL water.  Use 0.2-0.5 μL.
#*Dilute 1μL glycerol stock in 50μL water.  Use 0.2-0.5 μL.
-
#*Use 1pg-10ng of purified plasmid DNA
+
#*Use 1pg-10ng of purified plasmid DNA (volume < 0.5&mu;L)
#Heat sample to 95&deg;C for 3 mins to denature and cool to 4&deg;C.
#Heat sample to 95&deg;C for 3 mins to denature and cool to 4&deg;C.
#Mix 5 &mu;L reaction buffer with 0.2&mu;L enzyme mix for each reaction.  (Make up a master mix for multiple reactions).
#Mix 5 &mu;L reaction buffer with 0.2&mu;L enzyme mix for each reaction.  (Make up a master mix for multiple reactions).

Revision as of 13:32, 2 January 2007

< TempliPhi

Materials

Procedure

  1. Thaw sample buffer (white 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. Use 1μL to submit for sequencing. <-- This is what we used previously when sequencing on the Knight lab sequencer.
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