The plasmids used in this work, their main characteristics and construction details, are described in Table S1 . The DNA primers used in their construction are listed in Table S2 . Plasmids were constructed and purified using standard molecular biology procedures with proof-reading Phusion DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), DNA clean and concentrator™-5 kit and Zymoclean™ gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma Aldrich) or NZYMidiprep kit (NZYtech), according to the instructions of the manufacturers. The backbone plasmids used in this work were: pGADT7 (Clontech) and pGBKT7 (Clontech), used for yeast two-hybrid assays; pEGFP-C1 (Clontech), and pEF6/myc-His C (Thermo Fisher Scientific), used to generate mammalian transfection plasmids; and pSVP247 (da Cunha et al., 2017 (link)), a derivative of p2TK2—SW2 (Agaisse and Derre, 2013 (link)), used to generate a C. trachomatis expression plasmid bearing CT288 with a double hemagglutinin epitope tag (2HA) at its C-terminus (CT288-2HA). The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.
Geneelute plasmid miniprep kit
Manufactured by Merck Group
Sourced in Belgium
The GeneElute Plasmid Miniprep kit is a laboratory instrument designed for the rapid and efficient purification of plasmid DNA from bacterial cultures. The kit utilizes a silica-based membrane technology to capture and concentrate plasmid DNA, allowing for its isolation and purification.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (5 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (4 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (3 mentions)
Restriction enzyme, by Thermo Fisher Scientific (3 mentions)
Nzymidiprep kit, by NZYTech (3 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions)
Dna clean concentrator 5 kit, by Zymo Research (2 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Pentr d topo, by Thermo Fisher Scientific (1 mentions)
Advantage hd polymerase, by Takara Bio (1 mentions)
Vector nti, by Thermo Fisher Scientific (1 mentions)
Pcrii topo vector, by Thermo Fisher Scientific (1 mentions)
Bigdye v1, by Thermo Fisher Scientific (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Mix2seq on, by Eurofins (1 mentions)
Pmal c, by New England Biolabs (1 mentions)
Pgex 4t 2, by GE Healthcare (1 mentions)
15 protocols using geneelute plasmid miniprep kit
1
Plasmid Construction and Purification Protocols
2
Identifying Zebrafish NDST Homologs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse NDST protein sequences (Accession id:s NP_032332.2, NP_034941.2, NP_112463.2, NP_072087.1) were used as templates in tblastn searches of the Ensembl Danio rerio database (http://www.ensembl.org ). Hits with significantly lower E-values and higher sequence similarity to 3-O-sulfotransferase-2 than to any mouse NDST were regarded as non-relevant. Using this method, five putative ndst zebrafish transcripts were found; ndst1a; ENSDART00000085748, ndst1b; ENSDART00000090213, ndst2a; ENSDART00000125149, ndst2b; ENSDART00000085743, ndst3; ENSDART00000146084.
Primers were designed to amplify the sequences by PCR using Advantage HD polymerase (Clontech; Primer sequences are available upon request). PCR products were cloned into either pENTR/D-TOPO (Invitrogen) vector or pCRII-TOPO vector (Invitrogen). Plasmid DNA was purified with GeneElute plasmid miniprep kit (Sigma) and sequenced using Big Dye v1.1 (Applied Biosystems). Sequences from at least three separate PCR products of each gene were aligned and analyzed using VectorNTI (Invitrogen). Wherever there was a contradiction between the sequences, the “best of three-principle” was applied.
Primers were designed to amplify the sequences by PCR using Advantage HD polymerase (Clontech; Primer sequences are available upon request). PCR products were cloned into either pENTR/D-TOPO (Invitrogen) vector or pCRII-TOPO vector (Invitrogen). Plasmid DNA was purified with GeneElute plasmid miniprep kit (Sigma) and sequenced using Big Dye v1.1 (Applied Biosystems). Sequences from at least three separate PCR products of each gene were aligned and analyzed using VectorNTI (Invitrogen). Wherever there was a contradiction between the sequences, the “best of three-principle” was applied.
3
Optimized Plasmid and GTS Assembly
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All DNA fragments that constituted genetic elements used for plasmid or GTS assembly were amplified by polymerase chain reaction (PCR) using Phusion U Master Mix (ThermoFisher) according to the manufacturer’s recommendations. All PCR fragments used for cloning purposes were gel-purified using NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) by following the manufacturer’s protocol. All primers used in this study are denoted in Table_S1 and were purchased from Integrated DNA Technologies (IDT). GTSs with ∼400 bp upstream and ∼400 bp downstream targeting sequences for deletion of GOIs were constructed using the overlay PCR method ((26 (link)), see Supplementary Figure S1 for details). All plasmids were assembled by the uracil-specific excision reagent (USER™) method by cloning PCR fragments into AsiSI/Nb.bsmI cassette by following well-established protocols ((27 ), also see Supplementary Figures S2–S4 for details). Plasmid minipreps were prepared using GeneElute Plasmid MiniPrep Kit (Sigma-Aldrich) according to the manufacturer’s protocol. All newly constructed DNA constructs were validated by Sanger sequencing Mix2Seq ON (Eurofins). The full list of all plasmids used and constructed in this work is listed in Table_S2. All gene sequences were optimized using IDTs’ Codon Optimization Tool (https://eu.idtdna.com ).
4
Plasmid Construction and Purification for Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmids used in this work, their main characteristics and construction details, are described in S1 Table . The DNA primers used in their construction are listed in S2 Table . Plasmids were constructed and purified using standard molecular biology procedures with proof-reading Phusion DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), DNA clean & concentratorTM-5 kit and ZymocleanTM gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma Aldrich) or NZYMidiprep kit (NZYtech), according to the instructions of the manufacturers. The backbone plasmids used in this work were pGEX-4T-2 (GE Healthcare) and pMal-c (New England Biolabs), for recombinant protein purification, and pEGFP-C1 (Clontech) for transfection of mammalian cells. Furthermore, pSVP247 (S1 Fig and S1 Table ), a derivative of p2TK2-SW2 [28 (link)], was the backbone to generate C. trachomatis expression plasmids bearing genes whose transcription is halted by the incD terminator (TincD) and encoding proteins with a double hemagglutinin epitope tag (2HA) at their C-terminus. The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.
5
Constructing Chimeric Hxt11/2 Transporter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For PCR amplification, Phusion® High‐Fidelity PCR Master Mix with HF buffer was used (Finnzymes; Fisher Scientific, Landsmeer, the Netherlands). Restriction enzymes and T4 DNA ligase were acquired from Fermentas (Fisher Scientific). The open‐reading frames for HXT11 and HXT2 were PCR‐amplified from gDNA of strain DS68616 using primers F HXT11/R HXT11, and F HXT2/R HXT2 (Supplementary Table SII). To construct of chimeric HXT11/2, 1st loop of HXT11 and HXT2 was PCR‐amplified using F HXT11/R HXT2 TM1, and F HXT2 TM1/R HXT2. And the fragments were used as template DNA of chimeric HXT11/2 in an overlap PCR using the outside primers F HXT11 and R HXT2 and cloned into pRS313P7T7 using XbaI and Cfr9I.
The HXT11 and HXT2 PCR fragments were sequenced for validation, cut using restriction enzymes XbaI and Cfr9I (Fermentas) and cloned in yeast expression vector pRS313‐P7T7 behind the constitutive HXT7 (−391) promoter (P7) preceding the HXT7 terminator (T7). Plasmids were amplified and maintained in Escherichia coli DH5α cells. Plasmids were isolated from E. coli cultures using the GeneElute plasmid Miniprep kit (Sigma–Aldrich). The chimeric HXT11/2 and HXT2 expression construct were transformed into S. cerevisiae DS68625 using standard yeast genetic techniques (Gietz and Woods,2006 ).
The HXT11 and HXT2 PCR fragments were sequenced for validation, cut using restriction enzymes XbaI and Cfr9I (Fermentas) and cloned in yeast expression vector pRS313‐P7T7 behind the constitutive HXT7 (−391) promoter (P7) preceding the HXT7 terminator (T7). Plasmids were amplified and maintained in Escherichia coli DH5α cells. Plasmids were isolated from E. coli cultures using the GeneElute plasmid Miniprep kit (Sigma–Aldrich). The chimeric HXT11/2 and HXT2 expression construct were transformed into S. cerevisiae DS68625 using standard yeast genetic techniques (Gietz and Woods,
6
Plasmid Verification by Colony PCR and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Positive assemblies were confirmed by colony PCR using KAPA Taq PCR Kits (Kapa Biosystems) or Maxima Hot Start Green PCR Master Mix (Thermo Fisher Scientific) following the manufacturer’s protocols. For further verification, plasmids were isolated from positive colonies using GeneElute Plasmid Miniprep Kit (Sigma) or NucleoSpin Plasmid Easy Pure (Macherey-Nagel) and analysed by sequencing. DNA concentration and purity were evaluated using NanoDrop ND1000 spectrophotometer (Nanodrop technologies). Plasmid DNA and oligonucleotides were prepared according to service provider requirements (LGC Genomics or GATC service provider). Sequences were analysed with CLC Main Workbench (Qiagen). Primers used for colony PCR and sequencing are listed in S4 and S5 Tables.
7
Plasmid Transfection in 293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following DNA constructs were obtained from Addgene. First, pCMVHA EED wt (#24231), pCMVHA hEZH2 (#24230), pCMV-HA-JMJD3 (#24167), pCMV-HA-UTX (#24168) were gifts from Kristian Helin. Moreover, pcDNA3/Myc-DNMT1 (#36939), pcDNA3/Myc-DNMT3A (#35521), pcDNA3/Myc-DNMT3L (#35523) were gifts from Arthur Riggs. FH-TET1-pEF (#49792), and FH-TET3-pEF (#49446) were gifts from Anjana Rao. The isolation of the plasmid DNA from bacteria cultures was performed using Gene Elute Plasmid Miniprep Kit (Sigma; PLN70-1KT). For transfections, 293T cells were plated in a 12-well cell culture plate. Once the cells reached 80% confluency, they were transfected with 0.5 μg of DNA using Lipofectamine 3000 (Invitrogen; L3000015), according to the manufacturer’s instructions. The following day, the medium was aspirated from the plate and replaced with 250 μL/well of fresh supplemented EMEM. After 48 h, the cells were subjected to further analysis.
8
Plasmid Construction and Cloning Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids and oligonucleotides used in this work are listed in S4 and S5 Tables, respectively, as well as their relevant characteristics. Plasmids were generated using restriction enzymes or by restriction-free cloning [54 (link)]. For cloning using restriction enzymes, plasmids were constructed and purified using standard molecular biology procedures, using Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), NZYTaqII (NZYTech), DNA clean & concentrator™-5 kit, Zymoclean™ gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma-Aldrich) or NZYMidiprep kit (NZYTech) according to manufacturer’s instructions. For restriction-free cloning, plasmids were generated using a PCR-based method [54 (link)] with Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific), and DpnI (Thermo Fisher Scientific) was used to degrade parental plasmids. The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.
For the generation of plasmid-encoded Inc-2HA or Inc-GSK fusion proteins, inserts were composed of DNA sequences comprising the inc gene plus 300 base pairs upstream from the transcription start codon to include the promoter region.
For the generation of plasmid-encoded Inc-2HA or Inc-GSK fusion proteins, inserts were composed of DNA sequences comprising the inc gene plus 300 base pairs upstream from the transcription start codon to include the promoter region.
9
Plasmid Amplification and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR was performed with Phusion High-Fidelity Polymerase (Thermo Fisher Scientific, Waltham MA) or DreamTaq (Thermo Fisher) for cloning and diagnostic purposes, respectively. Oligonucleotide primers (SI Table S1) were purchased from Sigma-Aldrich. PCR products amplified from plasmid templates were digested with FastDigest DpnI (Thermo Fisher) to avoid contamination with template DNA. Fragment sizes were analysed by electrophoresis on 1% (w/v) agarose gels. PCR products were purified with the GeneElute PCR Clean-Up Kit (Sigma-Aldrich) or the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine CA). Plasmids were purified using with a GeneElute Plasmid Miniprep Kit (Sigma-Aldrich).
10
Cloning and Expression of HXT11 and HXT12
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For PCR amplification, Phusion® High-Fidelity PCR Master Mix with HF buffer was used (Finnzymes; Fisher Scientific, Landsmeer, The Netherlands). Restriction enzymes and T4 DNA ligase were acquired from Fermentas (Fisher Scientific, Landsmeer, The Netherlands). The open-reading frames for HXT11 and HXT12 were PCR-amplified from cDNA of the wild-type DS68625 using primers HXT11F (5′-GGCCTCTAGAATGTCAGGTGTTAATAATACATCCGC-3′), HXT12F (5′-GGCCTCTAGAATGGGTTTGATTGTCTCAATATTCAAC-3′) and HXT11•12R (5′-CGATGGATCCTCAG CTGGAAAAGA ACCTCTTGTA AATTG-3′). The HXT11 and HXT12 PCR fragments were sequenced for validation, cut using restriction enzymes XbaI and BamHI (Fermentas) and cloned in yeast expression vector pRS313-P7T7 behind the constitutive HXT7 (−391) promoter (P7) preceding the HXT7 terminator (T7). Plasmids were amplified and maintained in E. coli DH5α cells. Plasmids were isolated from E. coli cultures using the GeneElute plasmid Miniprep kit (Sigma-Aldrich, The Netherlands). The HXT11 and HXT12 expression construct were transformed into S. cerevisiae DS68625 using standard yeast genetic techniques [47 ].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!