Molecular biological methods

All solutions were autoclaved or sterile filtered before usage. All methods were adapted from “Molecular Cloning“(Sambrook J., 1989), if not stated elsewhere.

3.2.1.1 Plasmid DNA purification

5 ml or 100 ml LB medium, for small or large-scale plasmid DNA purification, respectively, were inoculated with a single E.coli colony harboring the plasmid of interest and grown to saturation over night at 37 °C. Cells were harvested and further processed according to instructions of the following plasmid purification kits: QIAprep spin miniprep Kit, QUIAGEN Plasmid Midi Kit and QIAprep Endofree Plasmid Maxi Kit (Quiagen) or Wizard®PlusSV Miniprep and PureYield™ Plasmid Midiprep system (Promega).

3.2.1.2 Determination of DNA concentration

DNA concentration of purified Plasmid DNA was measured by UV absorption spectroscopy at a wavelength of 260 nm. In H2O, a solution of 50 µg/ml of double-stranded

DNA exhibits approximately an absorbance A260 = 1. 3.2.1.3 Plasmid DNA sequencing

DNA sequencing was performed either by Medigenomix GmbH (Martinsried, Germany) or by Sequiserve (Vaterstetten, Germany).

3.2.1.4 DNA restriction digestion

DNA fragmentation for analysis and cloning was carried out by digesting plasmid DNA or PCR products with restriction endonucleases. Enzymes and supplied buffers were used as recommended by the manufacturer. 0.5-1 µg plasmid DNA was used for analysis and 8-15 µg plasmid DNA or 20 µl purified PCR product for cloning purposes. The reaction was incubated at the recommended temperature for 2-4 h. The restriction digest was incubated typically at 65°C for 20 min to heat-inactivate enzyme for further cloning or separated by agarose gel electrophoresis for DNA fragment analysis.

Material and Methods 42

3.2.1.5 Dephosphorylation of DNA fragments

Prior to ligation of DNA fragments carrying identical ends, 5’-end dephosphorylation was carried out in order to prevent self-ligation of plasmid DNA fragments. The removal of 5’ phosphate groups was catalysed by Shrimp alkaline phosphatase (SAP). 5 – 10 µg plasmid DNA were incubated with 1 U SAP and supplied buffer for 1 h at 37 °C. Subsequently, the reaction was heat inactivation by incubation at 65 °C for 15 min.

3.2.1.6 5’-DNA end overhand fill in

For generation of blunt ends, 3’-recessed ends were filled-in by Klenow fragment. 15 U Klenow fragment and 30 µM dNTP were added to a 100µl DNA restriction digest and incubated at 25˚C for 15 min. Klenow fragment was heat inactivated at 75˚C for 20 min.

3.2.1.7 DNA purification

Enzymatically modified DNA or PCR products were purified according to QIAquick PCR purification kit (Qiagen) or to Wizard®SV PCR clean-up system (Promega).

3.2.1.8 DNA agarose gel electrophoresis

For separation of DNA fragments or analysis of plasmid digests, DNA agarose gel electrophoresis was performed in TAE buffer and 1 – 2 % TAE-agarose gels, supplemented with 1 mg/ml ethidiumbromide, at 4 – 6 V/cm. Ethidiumbromide interchelates with DNA and can be visualized under UV light. A DNA mass standard was used for identification of DNA length. The DNA was loaded in loading buffer on agarose gel.

DNA sample buffer 30 % glycerol, 0.25 % bromphenol blue, 0.25 % xylencyanol FF TAE 40 mM Tris-acetate, 1 mM EDTA, pH 8.0

3.2.1.9 DNA extraction from agarose gels

After separation by agarose gel electrophoresis and staining, bands of interest were cut out and purified by Wizard®SV gel clean-up system (Promega) or QIAquick gel extraction kit (Quiagen) in order to isolate DNA fragments.

Material and Methods 43

3.2.1.10 Polymerase chain reaction (PCR)

For amplification of DNA fragments, the polymerase chain reaction (PCR) was performed according to the following protocol:

Table 8: Standard protocol for PCR.

Reaction mixture Reaction

0.25 µM primer 1 (sense) 0.25 µM primer 1 (antisense) 10 µl 10x Pfu polymerase buffer 10 µl DMSO

50 – 100 µg template DNA 250 µM dNTPs

1 – 2 U Pfu DNA polymerase II ddH20 to 100 µl final volume

1st cycle denaturation 95 °C 5 min 25 cycle denaturation 95 °C 30 sec annealing 60 °C 30 sec extension 72 °C 1 min/kb last cycle extension 72 °C 8 min 4 °C

3.2.1.11 Oligonucleotides annealing

100 pmol/µl sense and antisense oligonucleotides were mixed at a 1:1 ratio, incubated first for 3 min at 95 °C, following 10 min at 65 °C, and finally for 2 h at 37 °C. The annealed oligonucleotides were ligated into vector DNA

3.2.1.12 Site-directed mutagenesis

Site-directed mutagenesis was performed using the QuickChange™ System (Stratagene) following the manufacturers’ instructions. Vector DNA containing the gene of interest was amplified by PCR using a complementary set of sense and antisense primers, which introduced site-specific the desired mutation. A mutagenesis PCR reaction was typically performed using the following protocol:

Table 9: Standard protocol for site-directed mutagenesis.

Reaction mixture Reaction

125 ng primer 1 (sense) 125 ng primer 2 (antisense) 5 µl 10x Pfu polymerase buffer 5 – 50 ng template DNA 250 µM dNTPs

1 µl PfuTurbo™ DNA polymerase II ddH20 to 50 µl final volume

1st cycle denaturation 95 °C 30 sec 25 cycle denaturation 95 °C 30 sec annealing 55 °C 1 min extension 68 °C 2 min/kb last cycle extension 68 °C 20 min 4 °C ∞

Material and Methods 44 PCR products were treated with 1 µl endonuclease DpnI (20000U/ml) for1 hour at 37 °C to digest specifically methylated and hemimethylated parental template DNA. 1 – 5 µl of the reactions were transformed in E.coli XL1-Blue supercompetent cells and mutations were confirmed by DNA sequencing.

3.2.1.13 Ligation of DNA fragments

Ligation reaction was carried out to fuse compatible DNA ends. Usually vector and fragment DNA were mixed at 1:7 ratios with 0.5 U T4 DNA Ligase and supplied buffer. The reaction was incubated at 20 °C for 15 min in case of cohesive ends and for 2 h in case of blunt ends followed by transformation into chemical competent E.coli cells.

3.2.1.14 Preparation and transformation of competent E. coli cells

Chemical competent cells were prepared as described (Hanahan, 1983). 5 ml LB were inoculate with a single colony and grown to saturation at 37 °C. 2 ml of this pre-culture were used to inoculate 200 ml LB medium and grown to an OD600 of 0.3. Cells were chilled on ice

for 15 min and centrifuged at 1500 x g for 15 min at 4 °C. Pelleted cells were resuspended in 30 ml ice-cold TB1 and incubated on ice for 10 min. Then, cells were centrifuged again and resuspended in 4 ml ice-cold TB2. Aliquots were frozen in liquid nitrogen and stored at -80 °C. For transformation, 50 µl competent cells were mixed with 0.05 – 0.2 µg plasmid DNA or 1 – 5 µl ligation reaction and incubated on ice for 30 min. The cells were then heat shocked at 42 °C for 45 – 60 s and placed on ice for 2 min. After addition of 1 ml of LB medium, cells were incubated at 37 °C for 1 h with shaking. The cell suspension was subsequently plated on selective plates and incubated at 37 °C until colonies had developed.

TB1 100 mM RbCl, 250 mM MnCl2, 30 mM KAc, 10 mM CaCl2,

15 % glycerol, pH 5.8 (adjusted with 0.2 HOAc)

TB2 75 mM CaCl2, 10 mM RbCl2, 10 mM MOPS, 15 % glycerol, pH 6.5

Material and Methods 45