Genematrix basic dna purification kit

Genomic DNA was isolated, as described in the analysis of RH30 clones. It was used for the PCR reaction with AmpliTaq Gold™ 360 DNA Polymerase (Thermo Scientific) with primers overlapping the deletion region, which were the same used with clones screening. Bands with sizes around 500 bp for edited clones and 1200 bp or wild type (WT) were cut from the gel and purified with a GeneMATRIX BASIC DNA Purification Kit (EURx) according to the vendor’s protocol. Concentrations were measured with Nanodrop or Quawell Q5000. To amplify the purified product, the PCR reaction was performed with the same primers and AmpliTaq Gold™ 360 DNA Polymerase (Thermo Scientific). For sequencing, we used a BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Scientific) according to the vendor’s protocol and a 3500 Series Genetic Analyzer (Thermo Scientific).

The sgRNAs for PRODH/POX (CRISPR All-In-One Non-Viral Vector with spCas9) were ordered by ABM Company (Richmond, VA, Canada). The vector with expression construct (directed against PRODH1 isoform) was transformed into Escherichia coli DH5α and grown in Luria–Bertani (LB) media supplemented with 100 µg·mL⁻¹ ampicillin at room temperature for 24 h, as described previously [60 (link)]. The targeted plasmid was extracted by a plasmid DNA purification kit (Nucleobond Xtra Midi/Maxi, MACHERY-NAREL GmbH, Düren, Germany). After being precipitated by isopropanol, the purified samples were washed by 70% ethanol solution and then followed by a DNA cleaning-up step by a GeneMATRIX Basic DNA Purification Kit (EURX, E3545-01 protocol 1, Gdansk, Poland). The purified DNA concentration was estimated by NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA).

Isolation of genomic DNA from K. hansenii ATCC 53582 and plasmid DNA from E. coli TOP 10F bacterial strains as well as DNA purification from agarose gel were performed by solid phase extraction on silica spin columns (Gene MATRIX Bacterial and Yeast Genomic DNA Purification Kit and GeneMatrix Basic DNA Purification Kit, EURx, Gdansk, Poland).
ColorTaq Polymerase (EURx, Gdansk, Poland) and C1000 Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) were used in all PCR amplification procedures (preparation of insert with motAB genes, control over pTI99-motAB vector preparation and verification of K. hansenii transformation) with appropriate primers (Table S1) synthesized at Genomed Ltd., Warsaw, Poland.
pTI99-motAB vector preparation was done with standard methods (restriction hydrolysis with BamHI and HindIII enzymes (FastDigest, Thermo Fisher Scientific, Waltham, MA, USA), overnight ligation catalyzed by T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA) and transformation by a heat shock method of competent E. coli TOP 10F cells (prepared in-house).

The sgRNAs for PRODH/POX (CRISPR All-In-One Non-Viral Vector with spCas9) were ordered by ABM Company (Richmond, Canada). The vector with expression construct was transformed into Escherichia coli DH5α and grown in Luria–Bertani (LB) media supplemented with 100 µg·mL⁻¹ ampicillin at room temperature for 24 h. The targeted plasmid was extracted by a plasmid DNA purification kit (Nucleobond Xtra Midi/Maxi, MACHERY-NAREL GmbH, Düren, Germany). After being precipitated by isopropanol, the purified samples were washed by 70% ethanol solution then followed by DNA cleaning-up step by GeneMATRIX Basic DNA Purification Kit (EURX, E3545-01 protocol 1, Gdansk, Poland). The purified DNA concentration was estimated by NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA).

Coding sequence of laccase KbLcc1 was obtained using the OE-PCR method according to the protocol described by Heckman and Pease [30 (link)] and presented at Figure 2. First PCR reactions were performed using primers L1 LF/IntR to amplify exon 1 (product AB) and IntF/L1 LR to amplify exon 2 (product CD), with the conditions presented in Table 7 and the plasmid pJET1.2 with cloned KbLcc1 gene sequence used as a template. Products AB and CD were extracted from the agarose gel using agarose-out protocol from the GeneMatrix Basic DNA Purification Kit (EURx, Gdansk, Poland) and then used as the templates for PCR with primers L1 SF/L1 SR (Table 6) to obtain product AD (coding sequence of KbLcc1 laccase). Reaction conditions of PCR steps included the initial denaturation at 98 °C for 30 s; 25 cycles at 98 °C for 10 s denaturing, annealing at 64.5 °C for 30 s, and extension at 72 °C for 60 s; a final extension of 72 °C for 10 min followed by maintenance at 4 °C. Gel-purified product AD was cloned to pJET1.2/blunt Cloning Vector using CloneJET PCR Cloning Kit (ThermoFisher Scientific, Waltham, MA, USA) and the obtained vector was transformed into E. coli Top10 F’ and plated on LB medium with ampicillin (100 µg/mL).

The sgRNAs for PRODH/POX (CRISPR All-In-One Non-Viral Vector with spCas9) were products of ABM Company. The vector containing expression construct was transfected into Escherichia coli DH5α cultured in Luria-Bertani (LB) medium with 100 μg/ml ampicillin for 24 h at room temperature. The extraction of the targeted plasmid was performed using a plasmid DNA purification kit (Nucleobond Xtra Midi/Maxi, MACHERY-NAREL GmbH). The purified samples were precipitated with isopropanol, washed with 70% ethanol, and submitted to DNA cleaning-up step by the GeneMATRIX Basic DNA Purification Kit (EURX, E3545-01 protocol 1). DNA concentration was evaluated by NanoDrop™ 2000/2000c Spectrophotometer.