Lab Protocols

Electroporation

Dyalisis (with 0,0250 µm filter) for 20 minutes of 12µl of ligation solution and 12µl of digested plasmid.

Insert the electrocompetent cell into the electroporation machine, and electroporate at 250V . All is well f there is no spark and the time constant approximates 4,6.

PCR amplification

Miniprep: GenElute™ Plasmid Miniprep Kit

Bacterial cells are harvested via centrifugation, subjected to a modified alkaline-SDS lysis procedure and the DNA adsorbed onto silica in the presence of high salts. Contaminants are then removed by a simple wash step. Bound DNA is eluted in water or Tris-EDTA buffer.

Gel purification: GenElute™ Gel Extraction Kit

The GenElute Gel Extraction Kit combines silica-binding technology with the convenience of a spin or vacuum column format. DNA fragments of interest are extracted from slices of an agarose gel and are bound to a silica membrane. Contaminants are removed by a simple spin or vacuum wash. The bound DNA is then eluted.

The purified DNA is suitable for a variety of downstream applications, such as automated DNA sequencing, PCR, restriction digestion, cloning, and labeling.

Column purification: GenElute™ PCR Clean-Up Kit

The GenElute PCR Clean-Up Kit combines the advantages of silica binding with a microspin format. The DNA is bound on a silica membrane within the spin column. The bound DNA is washed and the clean, concentrated DNA is eluted in the buffer of choice. Each column can purify up to 100 μL or 10 μg of PCR amplified DNA and recover up to 95% of PCR products between 100 bp and 10 kb. More than 99% of the primers and most primer-dimers (<40 bp are removed).

PCR cloning: Clontech™ In-Fusion HD Cloning Plus

The In-Fusion Enzyme premix fuses PCR-generated sequences and linearized vectors efficiently and precisely, utilizing a 15 bp overlap at their ends. This 15 bp overlap can be engineered by designing custom primers for amplification of the desired sequences. This method can be used to clone single or multiple fragments into a single vector without subcloning.

Materials

The usual equipment was worn: gloves, glasses and coat (especially because of UV emission & Ethidium Bromide during electrophoresis). There's more information in the page related with Safety.

Methods

First, birth and growing of bacteria populations.

Secondly, insertion of the biobricks and plasmids chosen with E. Coli (Electroporation method, PCR Amplification, Electrophoresis).

Just after this step, bacteria selection (in function of the quantities od oligopeptids or phoA+prolin) and inclusion of a TA system in E. Coli/S. Cerevisae.

Then, addiction of fluorescent proteins (GFP, RFP) and determination of the quantities and the principal properties of our bacteria (including Emission Spectroscopy) and analize of their genetical sequences.

Finally, conservation of bacteria populations producting the desired molecules or proteins in good quantities, at cold temperature in petri boxes and bioreactor containers.

The Kits we used were detailed above in this page.