Difference between revisions of "Team:LMU-TUM Munich/Methods"

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Preparation of plasmid DNA

For transformation of E. coli XL-1 blue chemically competent cells, cells were thawed on ice for 5 mins. 50-100 ng plasmid DNA (or 200 ng ligation product) were added, and cells were further incubated on ice for 20-30 mins. Afterwards, a heatshock was applied at 42°C for 45 secs. Cells were then further incubated on ice for 2 mins, and 950 μl of LB medium were added. Cells were then shaken at 200 rpm for at least 1 h at 37 C, and plated on LB-agar plates containing the appropriate antibiotic. The next day, single clones were picked and used to inoculate a liquid culture of 5 ml LB medium. After being incubated overnight shaking (200 rpm) at 37°C, cells were spun down, and plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep Kit. If ligation products were used for transformation, analytical digestion of DNA was performed by digestion with suitable restriction enyzmes for 1 h at 37°C and the correct incorporation of the desired fragment was verified via analytical gel electrophoresis. Additionally, plasmids were sequenced using the Eurofins Genomics sequencing service.

DNA digestion and ligation

Agarose gel electrophoresis

Polymerase chain reaction

Mammalian cell culture

Luciferase assay for the quantification of expression levels