Knight:TempliPhi: Difference between revisions
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==Procedure== | ==Procedure== | ||
#Thaw sample buffer ( | #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. | #Transfer 5 μL of sample buffer to small PCR tube for each template to be amplified. | ||
#Add template to sample buffer | #Add template to sample buffer | ||
| Line 12: | Line 12: | ||
#*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. | ||
#Heat sample to 95°C for 3 mins and cool to 4°C. | #*Use 1pg-10ng of purified plasmid DNA (volume < 0.5μL) | ||
#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions) | #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. | #Transfer 5 μL TempliPhi premix to cooled sample. | ||
#Incubate at 30°C for 4-18 hours. | #Incubate at 30°C for 4-18 hours. | ||
| Line 19: | Line 20: | ||
#Heat to 65°C for 10 mins. | #Heat to 65°C for 10 mins. | ||
#Cool to 4 °C. | #Cool to 4 °C. | ||
#Use 1μL | #Send DNA for [[Knight:Sequencing DNA|sequencing]]. | ||
#* Use 2μL in 12μL for sequencing at the biopolymers facility | |||
#* Previously, for sequencing on the Knight lab sequencer, we used 1μL | |||
[[Category:Protocol]] | |||
[[Category:In vitro]] | |||
[[Category:DNA]] | |||
Latest revision as of 12:43, 18 November 2007
Materials
- illustra TempliPhi™ 100/500 Amplification Kit
- 0.6mL PCR tubes
Procedure
- 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
