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Team:Cambridge/Experiments/Initial Exercise Group control
From 2011.igem.org
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The picture below shows a map of the modified plasmid. | The picture below shows a map of the modified plasmid. | ||
[[File:cam_plasmid_positivecontrol.jpg | middle | thumb | 400px | map of the modified plasmid with mRUBY insertion]] | [[File:cam_plasmid_positivecontrol.jpg | middle | thumb | 400px | map of the modified plasmid with mRUBY insertion]] | ||
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| + | ===Experiment=== | ||
| + | The experiment involved the same steps as preparation and expression of gene fusions of the three teams. | ||
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| + | ====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 ([[Team:Cambridge/Protocols/PCR | protocol]] | ||
The control experiment consists of 3 reactions | The control experiment consists of 3 reactions | ||
Revision as of 10:01, 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.
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 control experiment consists of 3 reactions
- Reaction A
- 1μl primer ruby F
- 1μl primer ruby R
- 1μl mRuby (Elliot)
- Reaction B
- 1μl primer Vector F
- 1μl primer B reverse (provided)
- 1μl vector template
- Reaction C
- 1μl primer Vector R
- 1μl primer A forward (provided)
- 1μl vector template
