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Team:University of Lethbridge/Notebook/Project3July
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July 27, 2008
Nathan Phillips
Objective: Amplify TIR gene from E.coli total DNA from DH5a
PCR reaction setup:
-Enzyme = Phusion -Template = DH5a DNA -Buffer = HF Phusion buffer -Primers = IDT, designed & shipped July 16/08
PCR cycle conditions (programmed into HJ's thermocycler under "Nate":
1. Initial denaturation @ 95C for 3 mins (1 cycle) 2a. Denaturation @ 95C for 30 sec 2b. Annealing @ 49C 2c. Extension @ 72C for 30 sec (Repeat from step 2, 34 times) 3. Final extension @ 72C for 10 mins (1 cycle) 4. Hold at 4C
July 29, 2008
Nathan Puhl, Andrew, Alix
Objective: Optimize conditions for TIR PCR.
Set up PCR reaction for 5 reactions.
PCR cycle conditions:
1. Initial denaturation @ 98 C for 30 sec (1 cycle) 2a. Denaturation @ 98 C for 10 sec 2b. Annealing @ 42 C for 30 sec 2c. Extension @ 72 C for 15 sec (Repeat from step 2, 29 times) 3. Final extension @ 72 C for 7 mins (1 cycle) 4. Hold at 4 C
