Phire hot start 2 polymerase

The PCR amplification of the araR gene and its flanking regions for the sequencing experiments and that of the probes for the Southern and Northern blots was performed by using the Phire Hot Start II polymerase (Thermo Scientific, Breda, the Netherlands) according to instructions of the manufacturer using the Prime Thermal Cycler (Techne, (VWR) Amsterdam, the Netherlands) PCR machine. DNA isolation from the fungal mycelia frozen in liquid nitrogen was done according to the procedure described by Arentshorst et al. (2012 (link)) via a phenol-chloroform-isoamyl alcohol extraction. DNA extraction from agarose gels was performed using the GeneJET™ gel extraction kit from Fermentas Life Sciences (Amsterdam, the Netherlands) according to the manufacturer’s instructions. DNA concentrations were measured spectrophotometrically with the Thermo Scientific Nano Drop 2000 Spectrophotometer (Thermo Scientific, Breda, the Netherlands) at a wavelength of 260 nm. RNA extraction and Northern blot analysis were performed as described earlier (Alazi et al. 2016 (link)). Primers for the amplification of the probes are listed in Supplemental Table S1.

To analyse transcription of genes from the polycistronic messenger RNA of the ATNT16 P2A_P2A strain RNA was isolated using the MasterPure-Yeast RNA Purification Kit (Epicentre) from mycelium cultivated for 24 h in the absence or presence of 15 µg/ml doxycycline or pre-grown for 18 h without doxycycline and further cultivated for 6 h after addition of doxycycline. After a DNase treatment (TURBO DNase; ThermoFisher) RNA was transcribed into cDNA as previously described [19 (link)]. For normalisation of cDNA levels in the respective samples, serial dilutions were used for amplification of the A. niger actin gene using oligonucleotides 25 and 26. These primers span an intron region, which allows visualisation of a band shift from cDNA compared to genomic DNA (gDNA) and confirms the absence of contaminating gDNA in cDNA samples. For amplification of the melA gene oligonucleotides 15 and 16, for tyrP oligonucleotides 27 and 28 and for the tdTomato gene oligonucleotides 20 and 29 were used. PCRs of 30 cycles were performed in a SpeedCycler² (Analytik Jena) using Phire Hot Start II polymerase (Thermo Scientific).

After trypsinization, the ES cell pellets were lysed with 0.1 mg/ml proteinase K in lysis buffer (0.1 M Tris pH 8.5, 5 mM ethylenediaminetetraacetic acid, 0.2% sodium dodecyl sulfate [SDS], 0.2 M NaCl), and the DNA was isolated through ethanol precipitation. E6.5–E8.5 DNA was isolated using 40 μl of QuickExtract (Lucigen, WI) solution according to the manufacturer's instructions, and 1–5 μL of the lysate was used for PCR.
PCR was used to amplify a region in Nhlrc2, where the insertion of the targeting construct resulted in a new SacI digestion site, with the primers shown in Table 2. Phire Hot Start II Polymerase (Thermo Fisher Scientific, Waltham, MA) and a Piko Thermal Cycler (Thermo Fisher Scientific, Vantaa, Finland) were used according to the manufacturer's instructions. Sanger sequencing was performed to validate the PCR product as described previously (Hiltunen et al., 2020 (link)). The PCR product from E6.5–8.5 embryos was precipitated with NaCl (4 M, 1:10) and ethanol before digestion overnight at −20°C or for 30 min at −70°C. The PCR product was digested using the SacI restriction enzyme (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer's instructions and run on 1.5% agarose gel (BioNordika, Helsinki, Finland). SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA) was used for detection.

Primers for PCR and Sanger Sequencing are found in Table S3. The PCR reactions contained 200 nmol dNTPs, 1X Phire Hot Start II buffer, 1.25 pmol/µl primers and 0.2 µl Phire Hot Start II polymerase (Thermo Scientific) in 25 µl volume. Cycling conditions were 98°C 1.5 min, (98°C 30 s, annealing temperature 30 s, 72°C 30 s) for 30 cycles, 72°C 7 min. PCR products were cleaned using ZymoResearch DNA Clean and Concentrator. Sanger sequencing was performed by the VCU Nucleic Acids Research Facility or Eurofins Genomics (Louisville, KY).

DGGE analysis was used to visualise the population dynamics in both reactors over time. Bacterial 16S rRNA V6–V8 regions were amplified with the universal bacterial primers GC-968F and 1401R [24 (link)] using the Phire Hot start II Polymerase (Thermo Fisher Scientific, Waltham, MA). Bacterial amplicons were generated with a G-Storm cycler (G-storm, Essex, UK) using a pre-denaturing step at 95 °C for 5 min, followed by 35 cycles at 95 °C for 20 s, 56 °C for 40 s, 72 °C for 40 s and a post-elongation step of 10 min at 72 °C. The forward primer had a GC clamp of 40 bp attached to the 5′-end as used by Yu et al. [25 (link)]. DGGE analysis was performed as described by Martín et al. [26 (link)] in a DCode TM system (Bio-Rad Laboratories, Hercules, CA) at 60 °C for 16 h with a denaturing gradient of 30:60 percent gradients according to [25 (link)]. After electrophoresis, gels were silver-stained as described by Sanguinetti et al. [27 (link)] and scanned.

Extraction of genomic DNA was performed with the GenElute mammalian genomic DNA miniprep kit (Sigma Aldrich, MO, USA). For targeted integration PCRs Phire Hot Start II Polymerase (Thermo Fisher Scientific, MA, USA) was used under standard conditions. To detect the 5′-junction and 3′-junction of targeted integrated (TI) donor DNA and genomic DNA the following primers were used: 5′-TI_AAVS1_fwd: 5′-CCAGCTCCCATAGCTCAGTCTG-3′, 5′-TI_Puro_rev: 5′-GGTCCTTCGGGCACCTCGAC-3′, 3′-TI_AAVS1_rev: 5′-GGGCTCAGTCTGAAGAGCAGAG-3′, 3′-TI_CSF2RA^coop_fwd: 5′-GGGCAGCGTGTACATCTACG-3′. To discriminate between clones that were targeted mono-allelic or bi-allelic, the AAVS1_fwd and AAVS1_rev primers were used.