Team:University of Lethbridge/Notebook/Project3July

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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

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