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

• 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