Molecular methodology

2.2.1. Restriction mapping and subcloning

Restriction mapping, subcloning and plasmid DNA extraction from E. coli were carried out according to standard methods (Sambrook et al., 1989), and using the reagents according to the manufacturer´s instructions. E. coli competent cells were transformed with purified plasmids by the heat shock method described by (Hanahan, 1985). Restriction enzymes were provided by Roche (Barcelona, Spain). Ligations were carried out using T4 DNA ligase from Roche. DNA fragments were isolated from TAE electrophoresis gels using the QIAquick Gel extraction Kit (QIAGEN) following the manufacturer´s instructions or the “glass milk” extraction. For the latter DNA was cutting from a 1% of Low melting temperature agarose gel (NuSieveR GTGR Agarose/LONZA), transferred in a 2 ml Eppendorf centrifuge tube with 700 µl (3V) of NaI 6M buffer (1) and incubate 5 minutes at 55 °C. The tube was vortex during this incubation time until the agarose was completely dissolved. 15 µl of “glass milk” (2) solution was added and incubate for 10 minutes on ice with vortexing every 1minute. After centrifuging for 1 minute at full speed, the pellet was resuspended in 500 µl “new washing buffer” (3). The washing step was performed three times. After drying the pellet through an additional centrifuge step the pellet was resuspended in 20 µl ddH2O and incubated for 5

minutes at 55 °C. The mix was centrifuged for 1 minute at full speed and the supernatant containing the DNA was transferred to a new Eppendorf centrifuge tube.

(1) NaI 6M buffer: dissolve 90 g NaI; 1, 52 g Na2SO3 in 100 ml H2O, sterile filtrate and store at 4 ªC.

(2) Glass milk: 100 mg/ml silice powder (Sigma S5631) in PBS 1x (0,8 g NaCl; 0, 02 g KCl; 0,144 g Na2HPO4, adjust pH 7,4 add H2O until 100ml and autoclave)

30 wash with PBS (1x), let it sediment and resuspend in TE-Buffer (10 mM Tris, 1mM EDTA, pH 7,8).

(3) New washing buffer: 20 mM Tris pH 7,4; 1mM EDTA; 100 mM NaCl (1 ml 1M Tris pH 7,4; 100 µl 0,5 M EDTA; 1 ml 5 M NaCl; 47,9 ml H2O until all is

dissolved, then add 50 ml EtOH 100%), store at -20ºC

2.2.2. Nucleic acid (gDNA/RNA) extraction from F. oxysporum

During this work two different protocols to isolate genomic DNA from F. oxysporum were used. Genomic DNA was extracted from F. oxysporum mycelium using the CTAB method (Torres et al., 1993), to obtain a high concentration of clean DNA e.g for verification PCRs after single sporing of transformants or for Southern-blots. Briefly, approximately 100 mg of mycelium were ground to a fine powder in a mortar and pestle under liquid nitrogen and transferred to a 2 ml Eppendorf centrifuge tube with 1 ml of CTAB extraction buffer (1) and vortexed. 4 μl of β-mercaptoethanol (Merck) and 500 μl of a chloroform: octanol 24:1 (v/v) solution were added quickly, the mix was vortexed and incubated at 65ºC for 30 minutes. After incubating at room temperature for 15 minutes the tube was centrifuged for 5 minutes at 10000 g. The supernatant was then precipitated with 1 ml of 100% ice-cold ethanol and incubated at -20ºC for at least 10 minutes (optional over night), followed by centrifugation for 5 minutes at 7500 g and two consecutive washes with 1 ml 75 % ethanol. Finally, the pellet was resuspended in 50-100 μl of sterile deionised water with 4 μl de RNase (10 mg/ml) and incubated at 37°C for 30 minutes.

(1) CTAB extraction buffer: 12.1 g/l Trizma base; 7.44 g/l EDTA; 81.8 g/l NaCl y 20 g/l and 20 g/l Cetyltrimethylammonium bromide. Heat to 60 °C to dissolve and adjust to pH 8.0 with NaOH. Keep at 37 ºC to avoid precipitation.

The second DNA extraction method used in this work was the “Glass Beads” gDNA method which allows isolating gDNA from F. oxysporum mycelium growing on solid media. This method was used for fast DNA extraction from putative mutants growing on PDA masterplate after transformation. This DNA was exclusively used for a diagnostic PCR screening. Briefly, with a sterile spatula approximate 0, 5 mm2 of mycel were cut from the

31 plate transferred to a 2 ml Eppendorf centrifuge tube containing 500 µl Lysis Buffer (1). The mycel were homogenized with the T 10 basic ULTRA-TURRAX (IKA®) or optional mycel can be grounded manually with glass beads (0, 5 mm) and a spatula. After centrifugation at 7500 g for 2 minutes the supernatant was transferred to a new 2 ml Eppendorf centrifuge tube containing 275 µl 7M Ammonium acetate (2) and incubated 5 minutes at 65°C. After incubating 5 minutes on ice, 500 µl chloroform was added, the mix was vortexed and centrifuged at 7500 g for 3 minutes. The supernatant was transferred to a new Eppendorf centrifuge tube containing 1 ml Isopropanol and the DNA was precipitated by a 5 min incubation step at room temperature followed by centrifugation for 5 minutes at 7500 g and two washes with 1 ml 70 % ethanol. After drying the pellet was resupended in 20 µl deionised water with 4 μl de RNase (10 mg/ml) and incubated at 37ºC for 30-60 minutes. (1) Lysis Buffer : 100mM Tris pH 8.0; 50 mM EDTA; 1% SDS

(2) 7 M Ammonium acetate (NH4C2H3O2): 27 g Ammonium acetate in 50 ml deionised

water

For RNA extraction, approximately 100 mg of frozen mycelium were ground to a fine powder in a mortar and pestle under liquid nitrogen and transferred to a pre-chilled 2 ml Eppendorf centrifuge tube with 1 ml (4 volumes) of TRIzol Isolation Reagent (Life TechnologiesTM), followed by vortexing and incubation for 5 minutes on ice. After centrifugation for at 12000 g for 10 minutes at 4ºC the supernatant was transferred to a new vial. 200 μl of chloroform per 1 ml of TRIzol were added and the mix was vortexed for 15 seconds at low vortex power. After incubated on ice for 3 minutes the centrifugation at 4ºC for 20 minutes at 12000 g results in the formation of three phases. The upper clear phase with high-quality RNA was remove and transferred to a new clean 1, 5 ml Eppendorf centrifuge tube pre-chilled with 750 μl Isopropanol. The tube was mixed by inversion followed by incubation on ice for 10 minutes and centrifugation at 4°C for 20 minutes and 12000 g to precipitate RNA. The pellet was washed with 1 ml of 75% ethanol (v/v) and centrifuged at 4°C for 5 minutes and 7500 g. The pellet was air dried for 5-10 minutes and resuspended in 20-50 μl of RNase-free water.

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2.2.3. Nucleic acid quantification

DNA and RNA were quantified in a Nanodrop® ND-1000 spectrophotometer at 260nm and 280 nm wavelengths, respectively. In addition, the quality of the DNA and RNA obtained was monitored by electrophoresis in a 0.7% and 1% agarose gel (w/v), respectively.

2.2.4. DNA isolation form S. cerevisiae

For PCR analysis a fast DNA extraction was performed. A yeast cell was picked with a toothpick and transferred into a 1, 5 ml Eppendorf centrifuge tube with 50 µl of NaOH (0, 02 M). The tube was incubated for 20 minutes at room temperature during this time the tube was mixed several times. Then the Eppendorf centrifuge tube was cooked in a microwave for 2 minutes at maximal strength. The mix was spin down at 7000 rpm. 5 µl of the supernatant was used as a template for a PCR reaction with Vfin: 50 µl.

2.2.5. Plasmid isolation from S. cerevisiae cells

Plasmid isolation from Yeast cells was performed using the plasmid isolation kit (Roche). Briefly, a 5 ml over night culture of S. cerevisiae was centrifuged at 3000 rpm for 5 minutes. After discarding the supernatant the cell pellet was resuspended in 250 µl suspension buffer 1 (Roche) transferred to Eppendorf centrifuge tube and kept on ice. Add 2/3 volume glass beads (0, 5 mm) and shake 3x 20 seconds on a Mini BeadBeater-8 (BioSpec Products). Between these steps tubes were incubating on ice for 1 minute. After centrifugation at 13.200 rpm for 5 minutes at 4°C the supernatant was transferred to a clean Eppendorf centrifuge tube. 250 µl lysis buffer 2 was added and the tube was inverted gently 5 times and incubate for 2 min on ice. 350 µl chilled binding buffer 3 was added, mixed by gently invertion (5 times) and incubated on ice for 5 minutes. After centrifugation at 13.000 rpm for 10 minutes at 4°C the supernatant (700 µl) was apply to a column and centrifuged at 13.000 rpm for 1 minute at 4°C. The flow though was discarded and the column was washed with 700 µl washing buffer 5. After discarding washing buffer column was cleaned and dried by an additional centrifugation step for 1 minute. The plasmid was eluted by adding 30 µl elution

33 buffer 6 to the column and additional centrifugation as described before. 20 µl of the elution was used to transform competent E. coli cells.

2.2.6. Southern blot analysis

Southern analysis and probe labelling were carried out as described (Di Pietro and Roncero, 1998) using the non-isotopic digoxigenin labelling kit (Roche Diagnostics SL, Barcelona, Spain).