PCR amplification with Phusion Hot Start II high-fidelity polymerase (Thermo Scientific, Waltham, MA) was performed according to the manufacturer’s manual using high-performance liquid chromatography (HPLC)- or PAGE-purified, custom-synthesized oligonucleotide primers (Sigma-Aldrich). Diagnostic PCR was done with DreamTaq (Thermo Scientific) and desalted primers (Sigma-Aldrich). DNA fragments obtained by PCR were loaded on gels containing 1% or 2% (wt/vol) agarose (Thermo Scientific) and 1× Tris-acetate-EDTA buffer (Thermo Scientific), excised, and purified (Zymoclean, D2004; Zymo Research, Irvine, CA). Alternatively, fragments were purified using the GenElute PCR Cleanup kit (Sigma-Aldrich). Plasmids were isolated from E. coli with the Sigma GenElute Plasmid kit (Sigma-Aldrich) according to the supplier’s manual. Yeast plasmids were isolated according to the methods described in reference 50 (link). Yeast genomic DNA was isolated using a YeaStar genomic DNA kit (Zymo Research). E. coli DH5α (18258-012; Invitrogen) was transformed chemically (T3001; Zymo Research) or by electroporation. Chemical transformation was done according to the supplier’s instructions. Electroporation was done in a 2-mm cuvette (165-2086; Bio-Rad, Hercules, CA) by using a Gene PulserXcell electroporation system (Bio-Rad), following the manufacturer’s protocol.
Sigma genelute plasmid kit
Manufactured by Merck Group
The Sigma GenElute Plasmid kit is a laboratory product designed to extract and purify plasmid DNA from bacterial cultures. It provides a simple and efficient method for isolating plasmid DNA using a silica-based binding matrix.
Automatically generated - may contain errors
Lab products found in correlation
Genelute pcr clean up kit, by Merck Group (4 mentions)
Phusion hot start 2 high fidelity polymerase, by Thermo Fisher Scientific (4 mentions)
Dreamtaq polymerase, by Thermo Fisher Scientific (3 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (3 mentions)
Yeastar genomic dna kit, by Zymo Research (2 mentions)
Zymoprep yeast plasmid miniprep 2 kit, by Zymo Research (2 mentions)
Page purified oligonucleotide primers, by Merck Group (2 mentions)
Tae buffer, by Thermo Fisher Scientific (2 mentions)
Tris acetate edta buffer, by Thermo Fisher Scientific (2 mentions)
Desalted primers, by Merck Group (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Dreamtaq, by Thermo Fisher Scientific (1 mentions)
2 mm cuvette, by Bio-Rad (1 mentions)
Custom synthesized oligonucleotide primers, by Merck Group (1 mentions)
Gene pulser xcell electroporation system, by Bio-Rad (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide primer, by Merck Group (1 mentions)
Agarose gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using sigma genelute plasmid kit
1
PCR Amplification and Plasmid Isolation
2
Molecular Biology Techniques for Diagnostic PCR
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PCR reactions for diagnostic purposes were performed using DreamTaq DNA polymerase (Thermo Fisher Scientific, Walthman, MA, USA) according to manufacturer's instructions. When high fidelity amplification was needed, Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific) was used according to supplier's instructions. Oligonucleotides were ordered from Sigma Aldrich (St Louis, MO, USA) with PAGE or desalted purity depending on the purpose. DNA fragments were separated on agarose gels and were excised when purification of the fragment was required (Zymoclean, Zymo Research, Irvine, CA, USA). Bacterial plasmids were isolated using Sigma GenElute Plasmid kit (Sigma-Aldrich). When plasmid purification from yeast was required, Zymoprep Yeast Plasmid Miniprep II Kit was used (Zymo Research). Restriction digestion with DpnI for removal of circular templates (Thermo Fisher Scientific) was performed as recommended in the instruction manual. E. coli chemical transformations were performed following manufacturer's recommendations (Agilent Technologies).
Gene deletions were confirmed by diagnostic PCR and Sanger sequencing (Baseclear, Leiden, Netherlands).
Gene deletions were confirmed by diagnostic PCR and Sanger sequencing (Baseclear, Leiden, Netherlands).
3
Molecular Biology Experimental Techniques
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PCR amplification of DNA fragments with Phusion Hot Start II high-fidelity polymerase (Thermo Scientific, Waltham, MA) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) was performed according to the manufacturers’ instructions. DreamTaq polymerase (Thermo Scientific) was used for diagnostic PCR. Primers used in this study are shown in Table 4 . PCR products were separated by gel electrophoresis using 1% (wt/vol) agarose gels (Thermo Scientific) in Tris-acetate-EDTA (TAE) buffer (Thermo Scientific) at 100 V for 25 min and purified either with a GenElutePCR Clean-Up kit (Sigma-Aldrich) or with a Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA). Plasmids were purified from E. coli using a Sigma GenElute Plasmid kit (Sigma-Aldrich). Plasmids used in this study are shown in Table 5 . Yeast genomic DNA was isolated with the SDS-lithium acetate (LiAc) protocol (68 (link)). Yeast strains were transformed with the lithium acetate method (69 (link)). Four to eight single colonies were restreaked three consecutive times on selective media, and diagnostic PCRs were performed in order to verify their genotype. E. coli XL1-Blue was used for chemical transformation (70 (link)). Plasmids were then isolated and verified by either restriction analysis or by diagnostic PCR.
4
Molecular Cloning and DNA Analysis
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DNA fragments were amplified by PCR amplification with Phusion Hot Start II High Fidelity Polymerase (Thermo Scientific) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) performed according to the manufacturers’ instructions. Diagnostic PCRs were run with DreamTaq polymerase (Thermo Scientific). oligonucleotide primers used in this study are listed in Additional file 1 . PCR products were separated by electrophoresis on 1% (w/v) agarose gels (Thermo Scientific) in TAE buffer (Thermo Scientific) and, if required, purified with a Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA) or a GenElute PCR Clean-Up kit (Sigma-Aldrich). Yeast or E. coli plasmids were isolated with a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research), or a Sigma GenElute Plasmid kit (Sigma-Aldrich), respectively. A YeaStar Genomic DNA kit (Zymo Research) or an SDS/lithium acetate protocol [39 (link)] was used to isolate yeast genomic DNA. Yeast strains were transformed using the lithium acetate/polyethylene glycol method [40 (link)]. Single-colony isolates were obtained from three consecutive re-streaks on selective solid agar plates, followed by analytical PCR analysis of the relevant genotype. E. coli DH5α cultures were transformed by chemical transformation [41 (link)]. After isolation, plasmids were verified by restriction analysis and analytical PCR.
5
PCR Amplification and DNA Purification
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PCR amplification of DNA fragments with Phusion Hot Start II High Fidelity Polymerase (Thermo Scientific, Waltham, MA) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St Louis, MO) was performed according to manufacturers' instructions.
DreamTaq polymerase (Thermo Scientific) was used for diagnostic PCR. Primers used in this study are shown in Table 5. PCR products were separated by gel electrophoresis using 1 % (w/v) agarose gels (Thermo Scientific) in TAE buffer (Thermo Scientific) at 100 V for 25 min and purified with either GenElutePCR Clean-Up Kit (Sigma-Aldrich) or with Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Plasmids were purified from E. coli using a Sigma GenElute Plasmid Kit (Sigma Aldrich). Plasmids used in this study are shown in Table 4. Yeast genomic DNA was isolated with the SDS/LiAc protocol (66) (link). Yeast strains were transformed with the lithium acetate method (67) . Four to eight single colonies were restreaked three consecutive times on selective media and diagnostic PCR were performed in order to verify their genotype. E. coli XL1-blue was used for chemical transformation (68) .
Plasmids were then isolated and verified by either restriction analysis or by diagnostic PCR.
DreamTaq polymerase (Thermo Scientific) was used for diagnostic PCR. Primers used in this study are shown in Table 5. PCR products were separated by gel electrophoresis using 1 % (w/v) agarose gels (Thermo Scientific) in TAE buffer (Thermo Scientific) at 100 V for 25 min and purified with either GenElutePCR Clean-Up Kit (Sigma-Aldrich) or with Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Plasmids were purified from E. coli using a Sigma GenElute Plasmid Kit (Sigma Aldrich). Plasmids used in this study are shown in Table 4. Yeast genomic DNA was isolated with the SDS/LiAc protocol (66) (link). Yeast strains were transformed with the lithium acetate method (67) . Four to eight single colonies were restreaked three consecutive times on selective media and diagnostic PCR were performed in order to verify their genotype. E. coli XL1-blue was used for chemical transformation (68) .
Plasmids were then isolated and verified by either restriction analysis or by diagnostic PCR.
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