PCR amplification for cloning was performed with Phusion High Fidelity polymerase (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. DreamTaq polymerase (Thermo Fisher Scientific) was used for diagnostic PCR with yeast genomic DNA, isolated with the LiAc/SDS method (Lõoke et al. 2011 ) from overnight cultures on YPD, as template. Desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) are listed in Table S1 (Supporting Information ). PCR-amplified DNA fragments were analyzed by gel electrophoresis and, when required, purified from agarose gels with a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Prior to purification, template plasmid DNA was removed by FastDigest DpnI digestion (Thermo Fisher Scientific). Alternatively, DNA fragments were purified with a GenElute PCR Clean-Up Kit (Sigma-Aldrich). Gibson assembly with the NEBuilder HiFi DNA Assembly Master mix (New England Biolabs, Ipswich, MA), was performed with a down-scaled reaction volume of 5 µl and a total incubation time at 50°C of 1 h. The GenElute Plasmid Miniprep kit (Sigma-Aldrich) was used for plasmid isolation from overnight cultures of Escherichia coli XL1-Blue, which was used for plasmid amplification and storage.
Page purified oligonucleotide primers
Manufactured by Merck Group
Sourced in United States
PAGE-purified oligonucleotide primers are laboratory equipment used for the synthesis and purification of synthetic DNA or RNA oligonucleotides. The primers are produced through a process of polyacrylamide gel electrophoresis (PAGE) to ensure high purity and accuracy of the final product.
Automatically generated - may contain errors
Lab products found in correlation
Zymoclean gel dna recovery kit, by Zymo Research (6 mentions)
Genelute pcr clean up kit, by Merck Group (4 mentions)
Dreamtaq polymerase, by Thermo Fisher Scientific (4 mentions)
Genelute plasmid miniprep kit, by Merck Group (2 mentions)
Phusion high fidelity polymerase, by Thermo Fisher Scientific (2 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (2 mentions)
Phusion hot start 2 high fidelity polymerase, by Thermo Fisher Scientific (2 mentions)
Sigma genelute plasmid kit, by Merck Group (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Genelute plasmid miniprep kit, by Thermo Fisher Scientific (2 mentions)
Dreamtaq pcr master mix, by Thermo Fisher Scientific (2 mentions)
Tris acetate edta buffer, by Thermo Fisher Scientific (1 mentions)
Desalted primers, by Merck Group (1 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Fastdigest enzymes, by Thermo Fisher Scientific (1 mentions)
Topvision agarose, by Thermo Fisher Scientific (1 mentions)
Micropulser electroporator, by Bio-Rad (1 mentions)
Agarose gel, by Thermo Fisher Scientific (1 mentions)
Tae buffer, by Thermo Fisher Scientific (1 mentions)
6 protocols using page purified oligonucleotide primers
1
Cloning by PCR and Gibson Assembly
2
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.
3
Cloning and Plasmid Purification Protocol
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PCR amplification with Phusion High Fidelity Polymerase (Thermo Fisher Scientific) was performed according to the manufacturer's instructions using PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO, USA). Diagnostic PCR was done using DreamTaq polymerase (Thermo Fisher Scientific) and desalted primers (Sigma-Aldrich). All primer sequences are shown in Table 4 . DNA fragments obtained by PCR were separated by gel electrophoresis. Gel purification was carried out using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). PCR purification was performed using either the GenElute PCR Clean-Up Kit (Sigma-Aldrich) or GeneJET PCR purification kit (Thermo Fisher Scientific). Gibson assembly was done using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's recommendations. Restriction digest with PdmI and SmaI was performed using FastDigest enzymes (Thermo Fisher Scientific), according to the manufacturer's instructions. Escherichia coli strain XL1-blue was used for plasmid transformation, amplification and storage. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma-Aldrich).
4
Plasmid Construction via PCR and Yeast Transformation
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To amplify DNA fragments for plasmid construction, Phusion® High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA) was applied as specified in the manufacturer's protocol, using PAGE-purified oligonucleotide primers (Sigma-Aldrich). Diagnostic polymerase chain reaction (PCR) was performed with DreamTaq PCR Master Mix (Thermo Scientific), according to the manufacturer's protocol and with desalted oligonucleotide primers (Sigma-Aldrich). PCR-amplified linear integration cassettes were purified from 1% (w/v) agarose gels (TopVision Agarose, Thermo Fisher) with TAE buffer (50x, Thermo Fisher) using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). E. coli XL1-Blue competent cells were transformed by heat shock for 40 s at 42°C and, after 1 h recovery at 37°C in SOC medium, plated on selective LB ampicillin media. The GenElute Plasmid Miniprep kit (Thermo Fisher Scientific) was used to isolate plasmids from overnight cultures in 15 mL Greiner tubes on selective medium. S. cerevisiae was transformed with the lithium-acetate method (Gietz and Woods 2002 (link)). Transformants were selected on SMD agar with acetamide as sole nitrogen source. Single-cell lines of transformants were obtained by three consecutive re-streaks on solid selective medium.
5
Molecular Cloning and Transformation Protocols
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DNA fragments used for construction of plasmids and expression cassettes were amplified with Phusion high-fidelity DNA polymerase (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol and with PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO). Diagnostic PCR was performed with DreamTaq PCR master mix (Thermo Scientific) following the manufacturer’s protocol and with desalted oligonucleotide primers (Sigma-Aldrich). PCR-amplified linear integration cassettes were purified from 1% (wt/vol) agarose gels using a Zymoclean gel DNA recovery kit (Zymo Research, Irvine, CA, USA). E. coli DH5α was transformed by electroporation with a MicroPulser electroporator (Bio-Rad, Hercules, CA). Plasmids were isolated from overnight E. coli cultures on LB with ampicillin by the use of a GenElute plasmid miniprep kit (Thermo Scientific). Chemical transformation of S. cerevisiae was performed as described previously by Gietz and Woods (70 (link)).
6
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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