The pcDNA6 human wild-type (Insert size: 906 bp) and E46K mutant α-syn (Insert size: 1073 bp) plasmids were kindly provided by Prof. Hilal Lashuel (Addgene, Watertown, MA, USA; plasmid nos. 107425 and 105730) [58 (link)], while the enhanced green fluorescence protein (pEGFP-C1) control plasmid was obtained from Clontech Laboratories (BD Biosciences, Palo Alto, CA, USA; plasmid no. 6084-1). The DNA sequence of cloned inserts was next subjected to Sanger sequencing for confirmation at Apical Scientific Sdn. Bhd. (Selangor, Malaysia). All plasmid constructs were eventually purified with a Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA; Cat. #: A1330).
Wizard plus sv minipreps dna purification system
Manufactured by Promega
Sourced in United States, Germany
The Wizard Plus SV Minipreps DNA Purification System is a laboratory equipment used to isolate and purify plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to capture and elute DNA, providing a reliable and efficient method for DNA extraction.
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Lab products found in correlation
Pgem t easy vector, by Promega (20 mentions)
Wizard sv gel and pcr clean up system, by Promega (17 mentions)
T4 dna ligase, by New England Biolabs (10 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (5 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (5 mentions)
Qiaquick gel extraction kit, by Qiagen (5 mentions)
Smarter race 5 3 kit, by Takara Bio (5 mentions)
Taq dna polymerase, by New England Biolabs (4 mentions)
Mix and go e coli transformation kit, by Zymo Research (4 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (4 mentions)
Wizard genomic dna purification kit, by Promega (4 mentions)
Gotaq dna polymerase, by Promega (4 mentions)
Pgem t easy vector system, by Promega (4 mentions)
E coli jm109, by New England Biolabs (3 mentions)
Geneamp pcr system 9700 thermocycler, by Thermo Fisher Scientific (3 mentions)
Dna ligase, by Thermo Fisher Scientific (3 mentions)
Pgem t vector, by Promega (3 mentions)
Phusion dna polymerase, by New England Biolabs (3 mentions)
Dpni, by New England Biolabs (3 mentions)
Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (3 mentions)
149 protocols using wizard plus sv minipreps dna purification system
1
Plasmid Cloning and Sequencing Protocol
2
Cloning and Expression of P. gulae fimA Gene
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P. gulae genomic DNA was isolated as previously described [3 (link)] and used as a template for amplifying the entire fimA gene by PCR. The primers used for PCR were constructed from the sequences of the fimA genotypes [3 (link)] as follows: type A forward primers, 5′-GCG CGC GAA TTC GAG ATG AAA AAG ACT AAG-3′ and reverse primers, 5′-GCG CGG TTT AAG CTT TGA TTA CCA AGT AGC-3′; type B forward primers, 5′-GCG AAC GGA TCC AAG ATG AAA AAG ACT AAG-3′ and reverse primers, 5′-CGC TCT CTC GAG AGC TGA TTA CCA AAT-3; type C forward primers, 5′- ATC GAT ATC CAC TTT TAA AAC AAA AAA GAG-3′ and reverse primers 5′-TTT AGT CGT TTG ACG GGT CGA TTA CCA AGT-3′. Each amplified DNA was cloned into pGEM-T Easy Vector (Promega, Madison, WI), then digested with the appropriate restriction enzyme for ligation into the pET 42a (+) glutathione S-transferase (GST) gene fusion-protein expression vector (Novagen, Madison, WI, USA). The ligated vector was transformed into E. coli DH5α and colonies were selected on LB agar plates containing 50 µg of ampicillin/ml. Plasmid DNA was obtained using the Wizard® Plus SV Minipreps DNA Purification System (Promega). Each recombinant plasmid was transformed into E. coli BL21 (DE3) (Nippon Gene) and the colonies were selected as described above.
3
Methylation Profiling of Immune Checkpoint Genes
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The genomic DNA was extracted from tumor and normal tissues as described before and treated with bisulfite using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, United States). The bisulfite-treated DNA was then subjected to PCR for the amplification of the promoter regions of PD-1, CTLA-4, TIM-3, and LAG-3 using Hot Start TaKaRa Taq DNA Polymerase (TaKaRa Bio, Shiga, Japan). PCR primers were designed using MethPrimer software (http://www.urogene.org/methprimer/index1.html ), all are listed in Additional file 1 : Table S1b. The PCR products obtained were cloned into the pGemT-easy vector (Promega, Madison, USA) using DNA Ligation Kit, Mighty Mix (TaKaRa Bio). Ten individual clones from each sample were purified using Wizard® Plus SV Minipreps DNA Purification System (Promega) and sequenced with M13-reverse/forward primers (Additional file 1 : Table S1c). The promoter sequence details of immune checkpoints and PD-L1 genes are shown in Additional file 2 : Figure S2 A-F.
4
Cloning and Expression Cassette Construction
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The cloning vector harbouring the cDNA of cbhB was extracted from E. coli DH5 α cultured in 10 mL Luria-Bertani medium using the Wizard® Plus SV Minipreps DNA Purification System (Promega). To construct an expression cassette of the target gene, the plasmid from E. coli was digested with ClaI and KpnI (New England Biolabs, Beverly, MA) to release the insert, followed by gel purification of the target gene using the MEGAquick-spin™ Total DNA Fragment Purification kit (INTRON Biotechnology, Gyeonggi-do, South Korea) before it was inserted into the expression vector pPICzαC (Invitrogen/Life Technologies). Manipulation of DNA was carried out using standard procedures (Sambrook & Russell, 2001 ).
5
Cloning and Verification of DNA Fragments
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PCR amplifications of DNA fragments intended for cloning purposes was carried out using Q5 High-Fidelity DNA Polymerase (NEB, #M0491), whereas Taq DNA Polymerase (NEB, #M0273) was used for routine colony PCR. PCR amplified or restricted DNA fragments were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co, #740609). Plasmid DNA was purified using Wizard® Plus SV Minipreps DNA Purification System (Promega, #A1465). Vector and insert were mixed in 1:3 molar ratios (unless otherwise specified) and ligated in presence of T4 DNA ligase (NEB, #M0202) at 4°C for 18 h. For colony PCR, at least 10 randomly selected bacterial colonies were PCR amplified using a vector -specific and an insert -specific primer (Table 1 ). Plasmid DNA was isolated from positive transformants and verified using DNA restriction and sequence analyses.
6
Cloning and Characterization of LmTK Gene
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The LmTK gene (GeneDB LmjF.21.1210) was amplified using the PCR Extender System, from genomic DNA of L. major strain Friedlin with oligonucleotide primers 5´-CAT ATG TTC CGC GGT CGT ATA GAG-3´ and 5´-CTC GAG TCA CTC TGA GGA TGC AGC-3´ that include NdeI and XhoI restriction sites (in bold), respectively. The amplified full-length LmTK was cloned into the pGEMT vector and subsequently ligated into the NdeI and XhoI sites of pET28a vector. The resulting construct contained a His-tag at the N-terminus. Additionally LmTK was cloned into the expression vector pET22b, which carries a C-terminal His-tag sequence using the restriction sites of NdeI and HindIII. Plasmid DNA was isolated with the Wizard Plus SV Minipreps DNA Purification System (Promega). Positive clones were verified by genomic digestion and double-stranded DNA sequencing.
7
Cloning DNA Fragments via PCR and Transformation
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PCR amplifications of DNA fragments intended for cloning purposes was carried out using Q5® High-Fidelity DNA Polymerase (NEB, #M0491), whereas Taq DNA Polymerase (NEB, #M0273) was used for routine colony PCR. PCR amplified or restricted DNA fragments were purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co, #740609). Plasmid DNA was purified using Wizard® Plus SV Minipreps DNA Purification System (Promega, #A1465). Vector and insert were mixed in 1:3 molar ratios (unless otherwise specified) and ligated in the presence of T4 DNA ligase (NEB, #M0202) at 4oC for 18 h. 100 μl of E.coli DH5α as competent cells was transformed with 5 μl of vector-insert ligation mix and selected on LB agar plates containing 100 μg/mL ampicillin at 30°C overnight incubation. For colony PCR, at least ten randomly selected bacterial colonies were PCR amplified using a vector -specific and an insert -specific primer. Plasmid DNA was isolated from positive transformants that contained amplicon of expected size by PCR and then verified using DNA restriction.
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PCR amplifications of DNA fragments intended for cloning purposes was carried out using Q5® High-Fidelity DNA Polymerase (NEB, #M0491), whereas Taq DNA Polymerase (NEB, #M0273) was used for routine colony PCR. PCR amplified or restricted DNA fragments were purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co, #740609). Plasmid DNA was purified using Wizard® Plus SV Minipreps DNA Purification System (Promega, #A1465). Vector and insert were mixed in 1:3 molar ratios (unless otherwise specified) and ligated in the presence of T4 DNA ligase (NEB, #M0202) at 4oC for 18 h. 100 μl of E.coli DH5α as competent cells was transformed with 5 μl of vector-insert ligation mix and selected on LB agar plates containing 100 μg/mL ampicillin at 30°C overnight incubation. For colony PCR, at least ten randomly selected bacterial colonies were PCR amplified using a vector -specific and an insert -specific primer. Plasmid DNA was isolated from positive transformants that contained amplicon of expected size by PCR and then verified using DNA restriction.
8
Cloning and Sequencing PCR Products
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PCR products were excised from agarose gels and purified using the QIAquick Gel Extraction Kit (Qiagen). Products were cloned into ZERO Blunt PCR cloning vector (Invitrogen), transformed, and plasmid DNA recovered by mini-prep using the Wizard Plus SV Minipreps DNA Purification System (Promega). Individual clones were then sequenced. Sequence alignments were produced using MegAlign software from the Lasergene DNASTAR suite.
9
Plasmid-Based Mutation Controls for HRM
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To evaluate the specificity of HRM discrimination among different types of mutations, five plasmid-controls were designed and constructed. These plasmids contained four of the most predominant mutations (S531L, S531W, H526Y, or D516V) and a fifth wild-type (WT) plasmid with no mutation in the 81 bp hot-spot region. For each plasmid, 377 bp segment of rpoB gene containing the RRDR region was synthetically generated and then cloned into a 2507 bp pEX-K vector (Eurofins MWG Operon, Germany). All the vectors were then used to transform highly competent E. coli (New England Biolabs, MA, USA). Transformed E. coli cells carrying recombinant plasmid DNA were grown overnight in Luria Bertani (LB) broth (10 mL, Sigma Aldrich, USA) containing kanamycin (50 μg mL−1, Invitrogen, USA) at 37 °C at 300 rpm constant shaking. The bacterial cultures were harvested by centrifugation at 6000×g for 15 min at 4 °C. The supernatant was removed and the cell pellets consisting of the recombinant plasmid were isolated and purified with Wizard® Plus SV Minipreps DNA Purification System (Promega, USA) according to the manufacturer’s instructions. The purified plasmid DNA samples were quantified and then stored at −20 °C.
10
Sequencing and Characterization of M. oryzae dsRNA
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The dsRNAs extracted from M. oryzae strains from the Kyushu region, NHH12P-1, MZ4-12-2, and MZ13-12-1, were used as templates for cDNA synthesis using the PrimeScript™ first-strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan) with MoV2-specific primers (Supplementary Table S1 ). Three overlapping PCR products were amplified using the KOD FX Neo polymerase (Toyobo, Osaka, Japan) with the primer sets Hind-T7-MoV2F/MoV2-2697R, MoV2-2657F/MoV2-detect R 4052–4073, and MoV2-detect F 3703–3725/Bam-MoV2R-re (Supplementary Table S1 ). The amplified PCR products were treated with 10× A-attachment mix (Toyobo, Osaka, Japan) and ligated into the pGEM T-Easy vector (Promega, Madison, United States), which was then used to transformation Hit-DH5α cells (RBC Bioscience, Taipei, Taiwan). Plasmid DNA was purified using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, United States). Sequencing was conducted using both the universal primers (M13-M3 and M13-RV) and designed sequencing primers (Supplementary Table S1 ). The 5′- and 3′-terminal sequences of each dsRNA segment were determined using the SMARTer® RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, United States).
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