Seitenverantwortliche/r: Jan\

Basic techniques in molecular biology

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.

Determination of DNA concentrations

DNA concentrations were determined spectrophotometrically using a Nanodrop device. From the measured absorption at 260 nm, DNA concentrations were determined by assuming an absorption of 1 being equal to a concentration 50 ng/myl DNA. Furthermore, wavelengths at 280 nm and 230 nm were measured to confirm sample purity without contamination of proteins (280 nm) or carbohydrates, phenol or EDTA. Ratios of 260/280 nm being in the range between 1.6 and 2.0, as well as the ratios of 260/230 nm being between 2.0 and 2.2 generally indicate sample purity.

DNA digestion and ligation

For cloning, DNA was digested using restriction endonucleases. The amount of used enzyme hereby depended on the amount of to be digested DNA, whereas ~1 unit was used per myg of DNA. Buffers and DNA concentrations were used according to the manufacturer's suggestion. Digestions were incubated at 37°C. If deemed necessary, 5’-ends of vector fragments were dephosphorylated for 30 min at 37 °C directly by addition of alkaline phosphatase (Fast-AP) to the restriction mixture at 37°C.
Ligations were conducted using the appropriate DNA fragments while adding 400 units of ligase in the appropriate buffer. Vector and insert fragments were generally used at a 1:3 ratio, while the amount of vector fragment DNA was varied between 50-100 ng.

Agarose gel electrophoresis

Polymerase chain reaction

For the amplification of DNA fragments, polymerase chain reaction was conducted. Hereby, 50 µl PCR mixture contained 50-500 ng template DNA, 100 pmol of each primer, and 10 nmol of each desoxynucleotide (dATP, dCTP, dGTP, dTTP). Additionally, the mixture contained 1 U Q5 high-fidelity polymerase and Q5 polymerase buffer, and was filled up with ddH₂O to the final volume. For the PCR, double-stranded fragments were initially denaturated at 98 °C for 2 min and subsequently amplified in 25-30 repetitive cycles, each comprising three steps: (1) Denaturation of the double-stranded DNA (98 °C, 30 s), (2) annealing of the primer to the single-stranded template for 30 s at 50-65 °C (depending on the melting temperature of the primer-template hybrid), and (3) extension of the primed DNA template (72 °C, 60 s per kb fragment length). After that, a final elongation step (2 min, 72 °C) was included to ensure that remaining single-stranded template molecules are fully elongated.

Then, the PCR product was separated by agarose gel electrophoresis to check for its correct size and isolated from the gel as described or directly isolated from the PCR reaction mixture, respectively using the Qiagen Gel extraction and PCR purification kit.

Preparation of chemically competent E. coli cells

Basic techniques in protein biochemistry

Protein purification

Discontinous SDS polyacrylamide gel electrophoresis (SDS-PAGE)

For analysis of recombinant protein expression, discontinuous SDS-polyacrylamide gel electrophoresis was conducted. Therefore, samples were diluted with Laemmli buffer (to a final concentration of 1x Laemmli buffer) and incubated at 95°C for 5 min. For electrophoresis, currents of 300 mA were used, while voltage was initially set to X V and raised to X V after 10 mins. For staining, the gel was overlaid with staining solution and heated in the microwave. The staining solution was then replaced by XX.

Team:LMU-TUM Munich/Methods

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