Fungal genomic DNA was extracted using a modified SDS method (Yan et al., 2016b (link)). PCR amplification was performed using KOD High-Fidelity DNA Polymerase FX (TOYOBO, KEX-101). Purification of DNA fragments was done using a Gel Extraction Kit (Omega, D2500-02) or a Cycle Pure Kit (Omega, D6492-02). Homologous recombination for fragment ligation with the plasmid was performed using Exnase Multis (Vazyme, C113-01). Total RNA was extracted with Trizol (Invitrogen), and the PrimeScript RT Master Mix (TAKARA, RR036A) was used for cDNA synthesis. NANODROP ONE (Thermo scientific) was used for measuring concentrations and purity testing. In Southern blot assay, restriction enzymes (HindIII, XbaI, and SpeI) used for genomic DNA digestion were from New England Biolabs (United States). For probe preparation, the PCR amplified fragment was purified using Cycle Pure Kit (Omega, D6492-02), and labeled with digoxin using DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, 11745832910). Probe hybridization was performed using a DIG Probe Synthesis Kit (Roche, 11636090910) and detected by DIG Nucleic Acid Detection Kit (Roche, 11175 041910).
Exnase multis
Manufactured by Vazyme
Exnase Multis is a laboratory equipment product designed for nucleic acid extraction. It utilizes a proprietary enzyme blend to efficiently isolate DNA and RNA from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Dig high prime dna labeling and detection starter kit 1, by Roche (1 mentions)
Dig probe synthesis kit, by Roche (1 mentions)
Dig nucleic acid detection kit, by Roche (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Dpni, by Thermo Fisher Scientific (1 mentions)
Hifair 1st strand cdna synthesis supermix, by Yeasen (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
4 protocols using exnase multis
1
Fungal Genomic DNA Extraction and Analysis
2
Generating Genetically Modified Fungus Strains
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To obtain GFP-labeled VdaSm-GFP, the pNEO-olic GFP plasmid (stored in our laboratory) was transferred to VdaSm. G418 was used to select the transformants on PDA medium.
To generate the knockout plasmids, pKOVsmNoxB, pKOVsmPls1 and pKOVsmCrz1, upstream and downstream genomic sequences of these genes were amplified with the corresponding primers (Table S1 ). The paired sequences were inserted into a position flanking the hygromycin resistant cassette of the vector pGKO-HPT with the Exnase MultiS (Vazyme, Nanjing, China), and then the knock-out plasmids were transformed as previously described [35 (link)].
To produce the complemented strains, including VdΔnoxb/VdNoxB and VdΔpls1/VdPls1, GFP-fused VdNoxB and VdPls1 under the native promoter were introduced into VdΔnoxb and VdΔpls1, respectively. RFP-fused VdKar2 under the Tef promoter were introduced into VdΔnoxb/VdNoxB and VdΔpls1/VdPls1, to detect the location of VdNoxB and VdPls1, respectively. The fusion plasmids were constructed as previously described [22 (link)], and the primers are listed inTable S1 .
To generate the knockout plasmids, pKOVsmNoxB, pKOVsmPls1 and pKOVsmCrz1, upstream and downstream genomic sequences of these genes were amplified with the corresponding primers (
To produce the complemented strains, including VdΔnoxb/VdNoxB and VdΔpls1/VdPls1, GFP-fused VdNoxB and VdPls1 under the native promoter were introduced into VdΔnoxb and VdΔpls1, respectively. RFP-fused VdKar2 under the Tef promoter were introduced into VdΔnoxb/VdNoxB and VdΔpls1/VdPls1, to detect the location of VdNoxB and VdPls1, respectively. The fusion plasmids were constructed as previously described [22 (link)], and the primers are listed in
3
Molecular Cloning Operations Protocol
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Molecular cloning operations were all carried out according to the manufacturer’s protocols. Primers were ordered from GENEWIZ Biotech (Suzhou). Human cDNA was synthesized by using Hifair® 1st strand cDNA synthesis superMix (YEASON, 11141ES) and human RNA extracted from HEK293T cells by TRIzol reagent. PCR was performed using Phanta Max Super-Fidelity DNA polymerase (Vazyme, P505-d1) in 50 µL reactions. In vitro, homologous recombination was carried out using Exnase MultiS (Vazyme, C113-02) in 20 µL reactions (with ~120 ng linearized vector and ~60 ng insert). Digestion of template was performed using DpnI (Thermo, ER1701) in Tango buffer.
4
Engineered Operon for Fluorescent Protein Expression
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The pYB1a-PobR-eGFP-Cmr contains the PobR coding sequence (CDS), and an engineered operon consisting of a Promoter P pobA and two CDS, enhanced green uorescent protein (eGFP) and the chloramphenicol resistance (Cmr) genes. PobR coding sequence and P pobA were synthesized by Genescript. PobR coding sequence was codon optimized based on E. coli preference while P pobA was synthesis based on its original sequence in Acinetobacter baylyi. The synthesized PobR CDS was ampli ed by PCR using the primers PobR-Gibson-F and PobR-Gibson-R. A DNA fragment containing the ampicillin resistance gene and the P15A ori was also ampli ed using the primers pYB1a-Gibson-F and pYB1a-Gibson-R with the pYB1a-eGFP plasmid as a template. The two fragments were mixed in the presence of the Exnase Multis (vazyme), followed by transformation into E. coli DH5α competent cells. The generated pYB1a-PobR-eGFP was digested by XhoI and BglII, and then mixed with the XhoI-BglII digested PCR products ampli ed by Cmr-F and Cmr-R primers using the Cmr CDS as a template. After the ligation reaction of the DNA fragments and the transformation into E. coli, the pYB1a-PobR-eGFP-Cmr was constructed.
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