DNA methods

2.8.2.1 Plasmid isolation from E. coli and S. cerevisiae E. coli

Plasmids were isolated using either the QIAprep Spin Miniprep Kit, the HiSpeed Plasmid Midi Kit or the plasmid extraction protocol according to Birnboim and Doly (Birnboim & Doly, 1979). Plasmid extraction kits were used according to the manufacturer’s protocol. Plasmid extraction protocol according to Birnboim & Doly (1979) was applied to 50 ml LB E. coli cultures. Cells were grown over night at 37°C at 200 rpm and harvested by centrifugation at 4000 g for 15 min. The cell pellet was resuspended in 2 ml B&D I solution containing 10 µg freshly added RNase and incubated at RT for 5 min. Afterwards 2 ml B&D II solution were added and the sapmle mixed by vortexing and incubated for 5 min at RT. After addition of 2 ml B&D III solution, the sample was mixed by inverting the tube and centrifuged for 15 min at max rpm. The supernatant was transferred to a new tube and mixed with 7.5 ml isopropanol and incubated for 20 min at -20°C for DNA precipitation. The precipitated DNA was collected by centrifugation and washed with 5 ml of 70% ethanol. The residual ethanol was removed and the pellet was air dried. The DNA pellet was resuspended in 1-2 ml a. dest depending on its concentration.

S. cerevisiae

Plasmids were isolated using either the QIAprep Spin Miniprep Kit or the Smash and Grab protocol according to Hoffman and Winston (1987). Plasmid extraction kits were used according to the manufacturer’s protocol with minor changes. Additionally 0.3 g of glass beads (Ø 0.25-0.5 mm) were added to the P1 buffer in the tube to disrupted cell walls mechanically by vortexing.

The Smash and Grab protocol was applied to yeast cultures grown <16 h in 5-20 ml liquid medium and harvested by centrifugation for 3 min at 8000-12000 g. The cell pellet was resuspended in 0.5-1 ml H₂O and centrifuged again at 12000 g for 2 min. The supernatant was discarded and the cell pellet resuspended in residual liquid through vortexing. 200 μl lysis buffer, 200 μl phenol/chloroform solution and 0.3 g of glass beads (Ø 0.25-0.5 mm) were added and the sample was vortexed in a thermomixer (Eppendorf) for 8 min at 1,400 rpm at 4°C. The content was centrifuged for 15 min at 12000 g. Subsequently, 250 μl of the upper, aqueous layer were 55

transferred in a fresh reaction tube. DNA was precipitated with isopropanol to remove residual phenol.

2.8.2.2 Isolation of RNA and genomic DNA from S. macrospora

RNA isolation: RNA isolation was done according to Elleuche and Pöggeler (2009) with minor changes. The mycilium was harvested, dried and grounded in liquid nitrogen. About 1 g of powdered mycelium was mixed with 1 ml Trizol and centrifuged for 10 min at 13000 rpm and 4°C. The supernatant was transferred to a new tube and mixed with 200 μl chloroform. After centrifugation, the chloroform-generated upper phase was transferred to a new tube mixed with the same amount of isopropanol and after 10 min of incubation at RT centrifuged for 10 min at 13000 rpm and 4°C. The obtained pellet was dried under an outlet and resuspended in 120 μl RNA free H2O. Finally, the sample was incubated for 30 min at 1000 rpm and 60°C in a thermomixer (Eppendorf).

DNA extraction: gDNA isolation was done using phenol/chloroform extraction according to Lecellier and Silar (1994). The mycelium was harvested, transferred to a 2.0 ml reaction tube and frozen at -80°C for 30 min. The frozen pellet was taken up in 600 μl S. macrospora lysis buffer.

8-10 glass beads (Ø 2.85-34.45 mm) were added to the tube to (finally) mechanically disrupt the cell wall in a TissueLyser (Eppendorf) for 5 min at 30 Hz. The sample was mixed with 500 μl phenol, briefly vortexed and centrifuged at 13000 rpm for 10 min. The upper (aqueous) phase was transferred to another 2.0 ml reaction tube and mixed with 1 ml phenol/chloroform by vortexing. After another centrifugation step, the aqueous layer was transferred to a new 2.0 ml reaction tube and mixed with 1 ml chloroform followed by centrifugation. Again, the aqueous layer was transferred into a 1.5 ml reaction tube. The DNA was precipitated by adding isopropanol and incubation for 15 min at RT. After a final centrifugation step, the DNA pellet was dried under an outlet and resuspended in an appropriate amount of H2O.

2.8.2.3 PCR

Amplification of S. macrospora cDNA and gDNA as well as verification of strains via PCR or colony PCR on E. coli or S. cerevisiae plasmid DNA was achieved with HotStarTaq Master Mix Kit (Qiagen), Molzyme MolTaq polymerase (Molzym GmbH & Co. KG), Pfu polymerase

56

(Promega GmbH) or Phusion High-Fidelity DNA polymerase (New England Biolabs). All polymerases were used according to the manufacturer’s protocol.

2.8.2.4 Purification of amplified DNA Gel extraction

DNA extraction from agarose gels was done using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacture’s protocol.

Micro-dialysis membranes

Dialysis of nucleic acids after PCR amplification was conducted to get rid of salts present in reaction buffers. Nucleic acids were pipetted on membranes (Millipore, 0.02 μm pore size) floating on a. dest. in a petri dish and incubated for 10 min at RT. The desalted DNA was removed from the membranes via pipetting and used for further experiments.

.

2.8.2.5 cDNA synthesis

To ensure that the template RNA contained no remaining gDNA, it was treated with DNaseI according to the manufacturer’s protocol. Reverse transcription reaction was done with Transcriptor High Fidelity cDNA Synthesis kit according to the manufacturer’s recommendations. 2 μg of RNA were conducted as template for reverse transcriptase reaction.

Each reaction was made twice, once with and once without reverse transcriptase. The sample without template served after RNase treatment as control for final exclusion of gDNA remnants by PCR.

2.8.2.6 Hydrolysis of nucleic acids.

DNA for southern blot analysis, ligation after gel extraction or plasmid verification was done by hydrolysis with restriction enzymes according to the manufacturer’s protocol. The sample size depended on further use or experimental setup (generally ranging from 5-75 µl).

2.8.2.7 Ligation of DNA fragments

Fragments were ligated into the respective vectors using either QIAGEN PCR Cloning Kit (Qiagen), T4 ligase (Thermo Scientific) or In-Fusion® HD Cloning Kit (Clontech). QIAGEN 57

PCR Cloning Kit and Clontech In-Fusion® HD Cloning Kit were used according to the manufacture’s recommendations.

T4 ligase reactions were set up as follows: 1 μl T4 ligase, 2 μl 10x ligation buffer, 1-2 μl vector DNA and 15-16 μl dialyzed PCR fragment. The sample was incubated at 30 or 37°C for 30 min or overnight at 16°C. Before ligation with T4 ligase, the hydrolyzed vectors were dephosphorylated with calf intestine alkaline phosphatase (CIAP, Thermo Scientific). The general setup contained 2.5 μl 10x CIAP buffer, 1 μl CIAP and 21.5 μl vector DNA, with incubation at 37°C for 10 min followed by 10 min at 75°C to inactivate the enzyme.

.

2.8.2.8 Separation of nucleic acids by gel electrophoresis DNA

DNA fragments contained after PCR or digestion were mixed with 6x loading dye and separated in 0.8-1% agarose gels (1% [w/v] agarose in 1x TBE buffer) in a gel electrophoresis chamber at a voltage of 50-135 V. 0.5x TBE buffer was used as electrophoresis buffer. The gel, containing the separated DNA was incubation in a 1 μg/ml ethidium bromide solution for 10-20 min and visualized under UV light.

RNA

RNA fragments were separated in agarose gels containing 1.2% 1x MOPS and 5% formaldehyde at 80-100 V in a gel electrophoresis chamber. Prior, the respective RNA was mixed in a ratio 1:1 with 2x RNA loading dye already containing ethidium bromide and incubated for 10 min at 65°C. 1x MOPS was used as electrophoresis buffer. Similar to DNA, RNA was visualized under UV light, Gene Ruler DNA Ladder Mix served as marker.

2.8.2.9 Southern blotting

Total gDNA of the respective strains for Southern blotting was isolated as described above.

Depending on the final concentrations, about 25 µl of the obtained gDNA was hydrolyzed and separated by gel electrophoresis. The gel was washed in buffer I (0.25 M HCl) for 10 min, in buffer II (0.5 M NaOH) for 25 min and finally in buffer III (1.5 M NaCl, 0.5 Tris, pH 7.4) for 30 min. The DNA was transferred to a GeneScreen Hybridization Transfer Membrane (PerkinElmer) by capillary blotting technique and cross-linked to the membrane via exposure to

58

UV light for 5 min. DNA-DNA hybridization and detection reaction were done with High Prime DNA Labeling and Detection Starter Kit II (Roche) according to the manufacturer’s protocol.

After adding the detection agent to the membrane, the X-ray films were exposed for a minimum of 30 min to a maximum of 6 hours.