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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 | + | The three reactions performed ae the following: |
; '''Reaction A''' | ; '''Reaction A''' | ||
| - | :1μl primer ruby F | + | :1μl primer ruby F (provided) |
| - | :1μl primer ruby R | + | :1μl primer ruby R (provided) |
| - | :1μl mRuby | + | :1μl mRuby template |
; '''Reaction B''' | ; '''Reaction B''' | ||
| - | :1μl primer Vector F | + | :1μl primer Vector F (provided) |
:1μl primer B reverse (provided) | :1μl primer B reverse (provided) | ||
| - | :1μl | + | :1μl plasmid template |
; '''Reaction C''' | ; '''Reaction C''' | ||
| - | :1μl primer Vector R | + | :1μl primer Vector R (provided) |
:1μl primer A forward (provided) | :1μl primer A forward (provided) | ||
| - | :1μl | + | :1μl plasmid template |
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | ||
Revision as of 10:06, 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 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
