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Team:University of Lethbridge/Notebook/Project2July
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Objective: Amplify Riboswitch sequence from pTopp plasmid using PCR Reaction | Objective: Amplify Riboswitch sequence from pTopp plasmid using PCR Reaction | ||
Revision as of 17:56, 30 July 2008
Contents |
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.
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 -Lanes 2-4: 1:10 template dilution, annealing temps (45.0 C, 49.0 C, 53.0 C) - Lane 5-6: 1:100 template dilution, annealing temps (45.0 C, 49.0 C)
Gel #2: - Lane 1: 100 bp ladder - Lane 7: 1:100 template dilution, annealing temp (53.0 C) - Lane 8: negative control
Results? Bands present in all lanes! (all less than 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.
