Molecular biology methods

3.3.1. Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) was used for assembly and amplification of gene fragments and to screen for E. coli colonies transformed with desired plasmids (colony PCR).

A typical PCR mixture is shown in the table below:

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Component Volume (µl)

oligonucleotide primer up (10 µM) 1 oligonucleotide primer lo (10 µM) 1 Template (plasmid DNA) 0.5 5x Q5 reaction buffer 10

dNTPs (10 mM each) 1

Q5 High-Fidelity DNA Polymerase 0.25

ddH2O Ad 50 µl

PCR reactions with an extension of the desired sequence with two reverse primers, 10 cycles were performed with the first primer pair. After that, the second outer primer was added and another 30 cycles were performed. Single and small mutations were introduced by splicing by overlap extension (SOE) PCR. Two individual PCRs were performed and the mutation was inserted in one of the primers.

The second PCR included a primer that was complementary to a great part of the mutational primer. A third PCR ligates both fragments together with only the outer primer pair used. Colony PCR was performed using a small sample from E. coli colonies that was boiled in 15 µl of H2O for 10 min at 98 °C and 1 µl of this was used as template for the PCR reaction. If possible, 10 × Thermopol Reaction Buffer and TAQ DNA polymerase were used for colony PCRs.

A typical PCR procedure included an initial denaturation step for 2 min at 98 °C followed by 30 cycles with consecutive steps for denaturation (30 s at 98 °C), primer annealing (30 s at 52-57 °C) and elongation (30-90 s at 72 °C) as well as a final elongation step for 2 min at 72 °C.

3.3.2. Restriction digest

Restriction digests of PCR products and DNA plasmids were typically performed using 18 µL purified PCR product or 2 µg purified plasmids with 2 µL of 10× CutSmart buffer and 1 µL of each restriction enzyme. H2O was used to fill up to the final reaction volume of 20 µL. Reactions were usually incubated for at least 1 h at the recommended temperature. Samples including ApaI were first incubated for 1 h at 25 °C followed by the addition of further enzymes and another incubation step for 1 h at 37 °C.

3.3.3. DNA Ligation

After restriction digest of PCR fragments and plasmids with the appropriate enzymes, both fragments were ligated using T4 DNA ligase. Therefore 5 – 8 µl digested plasmid backbone and 30 – 40 µl of insert were mixed with the appropriate volume of 10x T4 DNA ligase buffer to a final 1x concentration and 1

Methods 43 µl of T4 DNA ligase. After a reaction at RT for at least 30 min the ligation was precipitated using ethanol-ammonium acetate precipitation (3.3.5) and used for transformation of E. coli DH5α.

3.3.4. Agarose gel electrophoresis

PCR reactions, restriction digests and plasmids were analyzed for their proper sizes using agarose gel electrophoresis. Therefore 1 % (w/v) agarose was dissolved in 1x TAE buffer by boiling the mixture shortly using a microwave oven with open lid. The liquid agarose was stored in a 60 °C chamber. Directly before running a gel, 5 ml HDGreen were added to 25 ml of agarose gel solution and poured into the appropriate chamber. Samples were prepared with 6x loading dye and transferred to the gel with an additional 2-log DNA ladder as length standard. The gel was run at 110 V for 20 – 40 min in 1x TAE buffer. Afterwards the gel was irradiated with UV light and analyzed on a GelDoc-It2 device with the 535 nm emission filter. When fragments needed to be cut out from a gel, it was placed on a UV-transilluminator and UV irradiation was strictly limited to define the edges of the fragments to minimize UV-mediated DNA damages like thymine dimers.

3.3.5. DNA purification

DNA from digestions and SOE PCR fragments were cut from agarose gels and isolated using Wizard SV Gel and PCR Clean-up System. Digested PCR products were directly purified via the same system without prior gel electrophoresis.

DNA from ligation mixtures was prepared for transformation by ethanol-ammonium acetate precipitation. Therefore, 1/10 vol. 7 M ammonium acetate solution and 3 vol. 99 % ethanol were added and the resulting mixture incubated for at least 1 h at -20 °C. After centrifugation for 30 min at 14,000

× g the supernatant was discarded and resulting DNA precipitate dried at 37 °C. Finally, DNA was dissolved in 20 µL H2O.

3.3.6. Plasmid DNA isolation

Plasmid DNA was isolated in small scales from E. coli overnight cultures using Wizard Plus SV Minipreps DNA Purification System according to the manufacturer’s instructions. For larger scales and transfection of mammalian cells, PureYield Plasmid Midiprep System was applied.

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3.3.7. Generation of electrocompetent

E. coli

Transformation of E. coli was performed by electroporation. To make the bacteria electrocompetent all salts had to be removed. Therefore 50 ml dYT were inoculated with 500 µl of an O/N culture of DH5α or BL21 (DE3) and grown at 37 °C until an optical density at 600 nm (OD600) of 0.5–0.7 was reached.

The cells were centrifuged at 4000 rpm and 4 °C for 12 min. The supernatant was removed and the pellet was dissolved in 30 ml ice-cold ddH2O, followed by another centrifugation step. This washing step was repeated with 20 ml and 10 ml water. The remaining cell pellet was dissolved in 500 µl ddH2O for direct use or in 10 % DMSO in ddH2O for cryocompetent cells for later use. Cryostocks were divided in aliquots of 100 µl and stored at –80 °C.

3.3.8. Transformation of

E. coli

Before electroporation took place, cuvettes with a gap of 2 mm were pre-cooled on ice. Electrocompetent cells were either used directly after generation or thawed from a cryostock on ice. For retransformations, typically 1 µl of plasmid solution was added to 100 µl of competent E. coli. When bacteria were transformed with ligated plasmids, the complete volume was added. The mixture was added to the cooled electroporation cuvette and incubated for 2 – 5 min. A pulse of 2.5 kV, 25 µF and a resistance of 200 Ω was applied to achieve uptake of plasmids. Directly after the pulse 1 ml of warm medium was added to the cells followed by a regeneration at 37 °C for 1 h. Afterwards 50 µl (retransformations) or the whole volume (cloning) were plated on an agar plate with ampicillin.

3.3.9. DNA sequencing

Cloned plasmids were verified by DNA sequencing performed by SEQLAB sequence laboratories (Göttingen). Samples with 12 µL of purified plasmid (700 – 1200 ng) were mixed with 3 µL primer stock solution (10 µM).