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