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Team:Cambridge/Experiments/Initial Exercise Group control
From 2011.igem.org
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| - | * 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]] | + | * 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
