For RNase L knockdown, DNA sequences for shRNAs that target RNase L mRNA or for a scramble shRNA were individually cloned into pLKO.1-TRC vector. HeLa cells were infected by lentiviral shRNAs to generate stable cell lines with RNase L knockdown. To overexpress RNase L, RIGI or PKR, the N-terminal FLAG-fused RNase L, RIGI or PKR DNA sequences were amplified by Phanta Super-Fidelity DNA Polymerase (Vazyme Biotech) from HeLa cells and cloned into the p23-phage vector. The plasmids were stably transfected into HeLa cells. To overexpress Flag-tagged RNase L nuclease-dead mutation (H672N), the N-terminal FLAG-fused RNase L nuclease-dead mutation (H672N) DNA sequence was created from the N-terminal FLAG-fused RNase L using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). To generate plasmids for protein expression and purification, target DNA sequences, including RIGI, MAD5, TLR3, PKR, NF90, OAS1, ADAR1-p150, STAU1, STAU2, DDX5 and DDX21, were amplified by Phanta Super-Fidelity DNA Polymerase (Vazyme Biotech) from HeLa cells and cloned into pET-28a vector. PKR or NF90 with its dsRBMs deletion was created from the WT PKR or NF90 respectively using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Primers for plasmid constructions were listed in Table S5. All constructs were confirmed by Sanger sequencing.
Phanta super fidelity dna polymerase
Manufactured by Vazyme
Sourced in China
Phanta Super-Fidelity DNA Polymerase is a high-fidelity DNA polymerase enzyme used for PCR amplification. It exhibits enhanced proofreading activity and fidelity, resulting in accurate DNA replication with a low error rate.
Automatically generated - may contain errors
Lab products found in correlation
Clonexpress 2 one step cloning kit, by Vazyme (6 mentions)
Clonexpress ultra one step cloning kit, by Vazyme (3 mentions)
T4 dna ligase, by Thermo Fisher Scientific (3 mentions)
Mut express 2 fast mutagenesis kit v2, by Vazyme (2 mentions)
Clonexpresstm 2 one step cloning kit, by Vazyme (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Ciprofloxacin standards, by MedChemExpress (2 mentions)
Easytaq pcr master mix dye plus, by Vazyme (2 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (2 mentions)
Exnase 2, by Vazyme (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Pmd18 t vector, by Takara Bio (2 mentions)
Chembiodraw ultra 14, by Revvity Signals Software (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Matchmaker gal4 two hybrid system 3, by Takara Bio (1 mentions)
Pfastbac1 vector, by Thermo Fisher Scientific (1 mentions)
Hiscript q rt supermix for qpcr, by Vazyme (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Cycle pure kit, by Omega Bio-Tek (1 mentions)
75 protocols using phanta super fidelity dna polymerase
1
Genetic Manipulation of RNase L and Related Proteins
2
Yeast Two-Hybrid, Pull-Down, and BiFC Assays for AtRAD51, AtRAD51C, and AtXRCC3
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To construct the vectors for yeast two-hybrid, pull-down and BiFC assays, full-length AtRAD51, AtRAD51C and AtXRCC3 cDNA were PCR-amplified using Phanta Super-Fidelity DNA polymerase (Vazyme Biotech Co., Ltd, China) and appropriate primers (S2 Table ).
For the Y2H assay, full-length AtRAD51 and AtXRCC3 cDNA were purified and ligated into pGADT7 pGBKT7 by NdeI and BamHI double-enzyme digestion, and full-length AtRAD51C cDNA was purified and ligated into pGADT7 and pGBKT7 by NdeI and EcoRI double-enzyme digestion.
For the BiFC assay, full-length AtRAD51 and AtXRCC3 cDNA was purified and ligated into pXY103, pXY104, pXY105 and pXY106 by BamHI and SalI double-enzyme digestion, and full-length AtRAD51C cDNA was purified and ligated into pXY103, pXY104, pXY105 and pXY106 by XbaI and SalI double-enzyme digestion.
For the pull down assay, full-length AtRAD51 and AtXRCC3 cDNA was purified and ligated into pET32a and pGEX-6P-1 by BamHI and SalI double-enzyme digestion and full-length AtRAD51C cDNA was purified and ligated into pET32a and pGEX-6P-1 by EcoRI and SalI double-enzyme digestion. All constructs were verified by DNA sequencing.
For the Y2H assay, full-length AtRAD51 and AtXRCC3 cDNA were purified and ligated into pGADT7 pGBKT7 by NdeI and BamHI double-enzyme digestion, and full-length AtRAD51C cDNA was purified and ligated into pGADT7 and pGBKT7 by NdeI and EcoRI double-enzyme digestion.
For the BiFC assay, full-length AtRAD51 and AtXRCC3 cDNA was purified and ligated into pXY103, pXY104, pXY105 and pXY106 by BamHI and SalI double-enzyme digestion, and full-length AtRAD51C cDNA was purified and ligated into pXY103, pXY104, pXY105 and pXY106 by XbaI and SalI double-enzyme digestion.
For the pull down assay, full-length AtRAD51 and AtXRCC3 cDNA was purified and ligated into pET32a and pGEX-6P-1 by BamHI and SalI double-enzyme digestion and full-length AtRAD51C cDNA was purified and ligated into pET32a and pGEX-6P-1 by EcoRI and SalI double-enzyme digestion. All constructs were verified by DNA sequencing.
3
Cloning and Transient Expression of MdWRKY75 in Apple
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The full coding DNA sequence (CDS) of MdWRKY75 (MD13G1122100) in apple was obtained by GDR (https://www.rosaceae.org/ ), and PCR amplification was conducted using Phanta Super-Fidelity DNA Polymerase (P501-d1, Vazyme Biotech Co. Ltd., China) and the primer sequences listed in Table S4 . The full CDS fragment of WRKY75 was inserted into pSAK277 vector under the control of the 35S promoter with EcoRI and XhoI. The recombinant expression vector WRKY75-pSAK277 was transformed into Agrobacterium tumefaciens (GV3101), it was cultured at 37 °C, and then collected, subsequently resuspended in a solution (included 10 mm MES, 10 mm MgCl2, and 200 μm acetosyringone) to a final optical density of 0.8 ~ 1.0 at OD600, and then incubated at room temperature for 3 − 4 h. The infiltration protocol and culture methods for transient expression were adapted from previously described methods [40 (link), 41 (link)]. The infected apples were placed at 23 °C for 3 days. All fruit samples were frozen in liquid nitrogen upon collection, and stored at − 80 °C.
4
Isolation and Sequencing of Turnip SUC Genes
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Twelve SUC gene sequences of B. rapa (BraSUCs) were searched from the Brassica database (http://brassicadb.org/brad/ ). Primers for the amplification of the full-length coding DNA sequence (CDS) of the turnip SUCs were designed by Oligo7 software based on the sequences of BraSUCs. The amplification primer sequences are listed in supplemental Table 1 . Polymerase chain reaction (PCR) was performed in a 50-μL reaction mixture containing 2 μg cDNA and 1 μL Phanta Super-Fidelity DNA Polymerase (Vazyme Biotech Co., China, Nanjing) under the following PCR conditions: 3 min at 95 °C for pre-denaturation, 35 cycles of amplification (30 s at 95 °C, 30 s at 56 °C, and 90 s at 72 °C), and a final extension at 72 °C for 7 min. A 1% agarose gel electrophoresis staining was used to confirm the size of the genes, and the EasyPure® Quick Gel Extraction kit was used for the purification and recovery of DNA from the agarose gel. The PCR products of the purification were cloned into the PMD18-T vectors (TaKaRa Biotechnology, Dalian, China) and sequenced by TsingKe (Tsingke Biological Technology Co., Beijing, China).
5
Phylogenetic Analysis of nCBP Orthologues
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A BLASTn search was performed using the AtnCBP (Accession number: AF028809 ) nucleotide sequence as a query against the S. tuberosum genome (Spud DB) and the Solanaceae Genomics Network (SGN) to retrieve nCBP homolog sequences in potato and N. benthamiana, respectively. StnCBP and NbnCBP were amplified with specific primers (Supplementary Table 1 ) from the potato cultivar Eshu 3 and N. benthamiana, respectively, using Phanta Super-Fidelity DNA polymerase (Vazyme, China). Then, the PCR amplicons were cloned into the pCE-Zero vector (Vazyme, China). Five colonies were randomly selected and sent for sequencing (Sangon Biotech, China). To analyze the phylogenetic relationships of eIF4E orthologues in S. tuberosum, Arabidopsis thaliana, Solanum lycopersicum, and Nicotiana benthamiana, we performed a BLASTp search against the NCBI and SGN databases using the amino acid sequences of potato eIF4E family members as queries. The phylogenetic tree was generated using MEGA5.2 with neighbor-joining tree and 1000 bootstraps. The nCBP orthologues in Oryza sativa and Zea mays were also obtained by a BLASTp against the NCBI database. Then, an amino acid sequence alignment of nCBP orthologues from the above species was performed by DNAMAN to analyze whether the nCBP orthologues are conserved across species.
6
Yeast Two-Hybrid Construct Generation
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The yeast two‐hybrid vectors pGBKT7 and pGADT7 were constructed according to the MatchMaker GAL4 Two‐Hybrid System 3 (Clontech, CA, USA) manufacturer's manual. The ORF of TaGID1 and TaNGR5 were cloned into the pGADT7 vector to obtain the pGADT7‐TaGID1 and pGADT7‐TaNGR5. The full‐length ORF of TaNGR5, RhtL1 and Rht1 was cloned into the pGBKT7 vector to obtain the pGBKT7‐TaNGR5, pGBKT7‐RhtL1 and pGBKT7‐Rht1. PCR was performed using Phanta Super‐Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China). Primers are listed in Table S16 .
7
Generating Gene Deletion Mutants in Magnaporthe oryzae
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The gene deletion mutants were generated using the standard one-step gene replacement strategy. First, two 1.0 kb of sequences flanking the targeted genes were PCR amplified with primer pairs (S2 Table ). Then, the resulting PCR products of MoDNM1, MoDNM2, MoDNM3, MoFIS1, and MoMDV1 were digested with restriction endonucleases and ligated with the HPH cassette released from pCX62. Finally, the completed inserts were sequenced. The 3.4 kb fragments, which contain the flanking sequences and hygromycin resistance cassette, were amplified and transformed into protoplasts of Guy11. Putative mutants were screened by PCR and confirmed by Southern blot analysis. The complement fragments, which contain the entire MoDNM1, MoFIS1, MoMDV1, MoDNM1K43A, MoDNM1T64G, MoDNM1G157V, MoDNM1D226A, MoDNM1F627A, MoDNM1F631A, MoDNM1F632A, MoDNM1ΔDYN and MoDNM1ΔGED genes with their native promoter regions, were amplified by PCR (Phanta Super-Fidelity DNA Polymerase, Vazyme Biotech Co., Nanjing, China) with primers (S2 Table ) and inserted into pYF11 (bleomycin resistance) to complement the respective mutant strains.
8
Purification of Heterotrimeric G Protein
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The human Gαi1, rat Gβ1 and bovine Gγ2 open reading frames were codon-optimized and synthesized by using GENEWIZ (Suzhou, China). The C-terminus of Gγ2 was fused to the N-terminus of Gαi1 containing G203A and A326S mutations by a 9 amino acid linker (GSAGSAGSA). The sequences encoding Gβ1 and the Gγ2-Gαi1 fusion protein were amplified using Phanta Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) and were separately subcloned into a modified pFastBac1 vector (Invitrogen, Cergy Pontoise, France), which contained an expression cassette for an 8×His tag and a codon-optimized maltose binding protein (MBP) tag followed by a Tobacco Etch Virus (TEV) protease recognition site upstream of the inserted fragments. In addition, single C to S point mutations were introduced at the C3 residue of Gαi1 and the C68 residue of Gγ2, which are lipid modification sites, to abolish membrane targeting27 ,28 (link),29 (link) and to allow for the purification of the G protein complex as soluble protein.
9
Molecular Biology Reagents Sourcing
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MI standard was purchased from Rhawn Chemical Technology Co., Ltd. (Shanghai, China). The other chemicals used in this study were purchased from Sigma-Aldrich (MO, United States). T4 Polynucleotide Kinase, T4 DNA ligase, and all restriction enzymes were purchased from Thermo Fisher Scientific (MA, United States). Phanta Super-Fidelity DNA Polymerase, ChamQ SYBR qPCR Master Mix, and HiScript Q RT SuperMix for qPCR were obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). The Plasmid Mini Kit I and Cycle Pure Kit used were purchased from Omega Bio-Tek (GA, United States). Synthesis of DNA oligonucleotide primers and Sanger sequencing were provided by Genewiz (Suzhou, China).
10
Sensitive Real-Time PCR Quantification
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Reaction mixtures contained of 5 × buffer (contained 2 mM MgSO4), 0.5 mM each of the dNTPs, 1 μM primers, 0.02U Phanta® Super Fidelity DNA Polymerase (Vazyme, Nanjing, China), and 0.5ul of the cDNA template was made from the SMARTer Kit. The thermal cycling program for the amplification was as follows: 95 °C for 3 min, 45 cycles of 95 °C for 30 s, 56 °C or 60 °C for 15 s, and 72 °C for 15 s followed by 72 °C for 7 min. The triplicate PCR products were mixed and visualized in a 1.5% agarose gels. The expression levels were visualized by a chemiluminescence system (Fusion Fx7, Fisher Biotech) and quantified by Quantity One software (Bio-Rad, CA, USA). Data were shown as means ± SEM (n = 3). The statistically significant differences between groups were accessed by paired Student’s t test using Graphpad Prism 5 software (GraphPad). Detailed sequences of the primers were shown in Additional file 1 : Table S1.
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