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Team:Cambridge/Experiments/Initial Exercise Group control
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Revision as of 10:55, 15 July 2011
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Contents |
Positive Control Experiment
Construct Design
In the positive control experiment we replaced the Green Fluorescent Protein coding sequence with a coding sequence for mRUBY, which is a Bright Monomeric Red Fluorescent Protein. The picture below shows a map of the modified plasmid. File:cam_plasmid_positivecontrol.jpg | frameless | thumb | 600px | map of the modified plasmid with mRUBY insertion]]
Experiment
The experiment involved the same steps as preparation and expression of gene fusions of the three teams.
PCR reaction
- We amplified the mRUBY coding sequence and two arms of the plasmid in a PCR reactions. First, we performed a real-time PCR with Taq polymerase, but as most samples were poorly amplified, we decided to repeat the reaction with Phusion polymerase (protocol)
The three reactions performed ae the following:
- Reaction A
- 1μl primer ruby F (provided)
- 1μl primer ruby R (provided)
- 1μl mRuby template
- Reaction B
- 1μl primer Vector F (provided)
- 1μl primer B reverse (provided)
- 1μl plasmid template
- Reaction C
- 1μl primer Vector R (provided)
- 1μl primer A forward (provided)
- 1μl plasmid template
- The graph presents accumulation of product with time in real-time PCR:
