Biology - iGEM Bordeaux 2012

iGEM - Bordeaux - Material & methods

DNA is digested with Thermo Scientific Fast digest enzymes, according to the instructions given. In each reaction 500ng of DNA are digested with 1U of each enzymes needed and 2μL of 10X buffer. The reactions are performed in 20μL. To avoid star activity or unwanted cut, enzymes are heat inactivated by heating at 80°C for 5minutes.

The ligations are performed like described for a proper use of biobricks (here). However, we bring some modifications to the initial protocol. The DNA fragments are added in equimolar amounts ie 2μL of the digestion mix (50ng of DNA). The reaction is carried out in a final volume of 20μL containing: 6μL of the different digestion mix (backbone + two fragments), 2μL of 10X buffer, 2U of ligase and water. The mix is incubated at 22°C for at least 10 minutes (up to an hour). We performed a step of heat inactivation of T4 DNA ligase at 70°C for 5 minutes in order to improve the electrotransformation efficiency.

During the project process, we used electrocompetent DH5α strain of E. coli mainly, after trying DH10B. Both strains were tested with heat shock and electroporation, but we found more convenient to work this way.
The electroporations are made with 50μL of competent cells added with 5μL of ligation mix and 50μL of water in a standard micro-electroporation chamber. All the material is cooled to 4°C before the operation. The electroporation is done under usual conditions 2500 V, 200 Ω, 25μF. Bacteria are left 1hour in 100μL of SOB media and then 200μL are plated with the right antibiotic (see table 1) and grown up for about 14 hours at 37°C.

Table 1: antibiotic concentration for LB media.
Chloramphenicol Ampicillin Kanamycin Tetracycline
34μg/mL 100μg/mL 50μg/mL 5μg/mL

The next day, single colonies are used to start a small culture of 5mL in order to extract the recombinant DNA. DNA purification is performed with the Thermo Scientific GeneJET Plasmid Miniprep Kit.

Considering that our project demands a lot of assembly, we also made our amplified backbones by PCR using the protocol available on the iGEM site: here