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Team:Cambridge/Experiments/Initial Exercise Group control
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* The graph presents accumulation of products with time in real-time PCR: | * The graph presents accumulation of products with time in real-time PCR: | ||
[[File:cam_PCR_graph_positive_control.jpg | left | thumb | 400px | progress of PCR reaction]] | [[File:cam_PCR_graph_positive_control.jpg | left | thumb | 400px | progress of PCR reaction]] | ||
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====Gel Electrophoresis==== | ====Gel Electrophoresis==== | ||
| + | * Products of PCR reaction with Phusion polymerase were separated on 1% agarose gel according to the [[Team:Cambridge/Protocols/Gel_Electrophoresis | protocol]]. The location of bands corresponded to the rough estimate of predicted sizes of DNA fragments. | ||
====Gel Extraction of DNA==== | ====Gel Extraction of DNA==== | ||
| + | * We followed the [[Team:Cambridge/Protocols/Gel_Extraction_of_DNA | protocol]] to extract and purify DNA. | ||
====Gibson Assembly==== | ====Gibson Assembly==== | ||
| + | * We performed [[Team:Cambridge/Protocols/Gibson_Assembly | Gibson Assembly]] in order to obtain a complete plasmid with GFP gene replaced by mRUBY. | ||
====Transformation of ''E.coli'' Competent Cells==== | ====Transformation of ''E.coli'' Competent Cells==== | ||
| - | + | * We [[Team:Cambridge/Protocols/Transformation_of_E.Coli | transformed]] competent E.coli cells with products of the Gibson Assembly, and after an overnight incubation at 37°C we examined colonies under the fluorescent microscope. We could see around 20 colonies on the plates, each emitting bright red light. | |
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====Digestion with Restriction Enzymes==== | ====Digestion with Restriction Enzymes==== | ||
Revision as of 11:19, 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 products with time in real-time PCR:
Gel Electrophoresis
- Products of PCR reaction with Phusion polymerase were separated on 1% agarose gel according to the protocol. The location of bands corresponded to the rough estimate of predicted sizes of DNA fragments.
Gel Extraction of DNA
- We followed the protocol to extract and purify DNA.
Gibson Assembly
- We performed Gibson Assembly in order to obtain a complete plasmid with GFP gene replaced by mRUBY.
Transformation of E.coli Competent Cells
- We transformed competent E.coli cells with products of the Gibson Assembly, and after an overnight incubation at 37°C we examined colonies under the fluorescent microscope. We could see around 20 colonies on the plates, each emitting bright red light.
Digestion with Restriction Enzymes
Transformation of Bacillus subtilis Competent Cells
