Team:Cambridge/Experiments/Initial Exercise Group control

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:

progress of PCR reaction

Gel Electrophoresis

Gel Extraction of DNA

Gibson Assembly

Transformation of E.coli Competent Cells

Examination under Fluorescent Microscope

Digestion with Restriction Enzymes

Transformation of Bacillus subtilis Competent Cells