Team:University of Lethbridge/Notebook/Project3July

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===July 27, 2008===
===July 27, 2008===

Latest revision as of 16:37, 25 August 2008

Back to The University of Lethbridge Main Notebook

Contents

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
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