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Team:University of Lethbridge/Notebook/Project2July
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July 28, 2008
Nathan Puhl, Roxanne
Objective: Amplify Riboswitch sequence from pTopp plasmid using PCR Reaction
PCR reaction setup:
-Enzyme = Phusion -Template = pTopp plasmid, dilution series of 1:10, 1:100 and (-) control -Buffer = HF Phusion buffer -Primers = IDT, designed & shipped July 16/08 -Total # of reactions = 20
PCR cycle conditions (programmed into Brent's PCR machine under "iGEM":
1. Initial denaturation @ 98 C for 30 sec (1 cycle)
2a. Denaturation @ 98 C for 30 sec (35 cycles for step #2)
2b. Annealing via gradient for 15 sec (45.0 C to 53.0 C)
2c. Extension @ 72 C for 30 sec
3. Final extension @ 72 C for 7 mins (1 cycle)
4. Hold at 4 C
July 29, 2008
Roxanne, Selina
Objective: Visualize whether any Ribswitch PCR reactions (July 28) were successful. Expect to see a band around 75 bp.
Ran 1% TAE agarose gel at 100 V for 30 mins
Loaded 5 uL sample, 1 uL ladder
Samples:
Gel #1: - Lane 1: 100 bp ladder - Lane 2: Neg cntl - Lane 3: 1:10 template dilution, annealing temp (45.0 C) - Lane 4: 1:100 template dilution, annealing temp (45.0 C) - Lane 5: 1:10 template dilution, annealing temp (49.0 C) - Lane 6: 1:100 template dilution, anneling tenp (49.0 C)
Gel #2: - Lane 1: 100 bp ladder - Lane 2: 1:10 template dilution, annealing temp (53.0 C) - Lane 3: 1:100 template dilution, annealing temp (53.0 C)
Results? Bands present in all lanes! (all around 100 bp) However, unsure if the bands are primer bands or Riboswitch sequence DNA bands. Nathan to run a 3% Agarose gel tonight to try and get better resolution.
note: Double bands present in this gel as well. Two bands at ~1kb and 1.5 kb are present in lanes L10 and H100.
