Restriction enzymes were purchased from NEB Inc. (Massachusetts, USA). Primer oligonucleotides were commercially synthesized by GENERAL BIOL (Nanjing, China). Cellular RNA from HEK293T cells, A549 cells and DF-1 cells was extracted by lysing the cells with TRIzol reagent (R0016, Beyotime). The RNA was reverse transcribed into single-stranded DNA using RevertAid First Strand cDNA Synthesis Kit (K1622; Thermo Fisher). Full-length DNA fragments encoding DExD/H-box protein family members, RNA-binding proteins and mitochondrial anchor proteins were amplified by PCR and cloned into the protein expression vector pCAGGS [49 (link)] To facilitate western blot detection, HA-, MYC-, or FLAG-tag was added to some gene fragments. For coimmunoprecipitation analysis, the coding regions of the viral PB2, PB1, PA, NP, M1, M2, NS1 and NS2 proteins from influenza virus A/WSN/33 (H1N1) or A/Anhui/1/2013 (H7N9) were cloned into the expression vector pCAGGS in-frame with a FLAG-tag sequence at either the 5’ or 3’ end. Plasmids encoding mutant proteins were constructed by using a site-directed mutagenesis kit (Beyotime, China). All plasmid constructs were verified by Sanger sequencing. All primers and sequences used in this study are listed in S3 Table in the supplemental material.
Site directed mutagenesis kit
Manufactured by Beyotime
Sourced in China
The site-directed mutagenesis kit is a laboratory tool used to introduce specific modifications or mutations into DNA sequences. It enables targeted changes to be made within a gene or plasmid, allowing researchers to study the effects of these alterations on gene function or protein structure and behavior.
Automatically generated - may contain errors
Lab products found in correlation
Pmirglo vector, by Promega (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Trizol reagent, by Beyotime (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Seamless cloning kit, by Beyotime (1 mentions)
Dual luciferase reporter gene assay kit, by Beyotime (1 mentions)
Orion microplate luminometer, by Berthold Technologies (1 mentions)
Pmirglo, by Promega (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
Firefly luciferase reporter gene assay kit, by Beyotime (1 mentions)
Calcium phosphate cell transfection kit, by Beyotime (1 mentions)
Pmirglo dual luciferase vector, by Promega (1 mentions)
Escherichia coli bl21 de3, by Transgene (1 mentions)
Ni nta column, by GE Healthcare (1 mentions)
Pmirglo dual luciferase mirna target expression vector pmirglo vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
16 protocols using site directed mutagenesis kit
1
Molecular Cloning and Expression of Viral and Cellular Proteins
2
Validating lncRNA RPSAP52/STAT3 Binding
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The validation of the target was performed via luciferase reporter assay. The mutant type of lncRNA RPSAP52/STAT3 harboring the binding site was generated using site-directed mutagenesis kit (Beyotime, Shanghai, China). The reporter plasmid was constructed via cloning the wild type (WT) lncRNA RPSAP52/STAT3 or mutant type (MUT) RPSAP52/STAT3 binding sequence into pmirGLO vector (Promega, Madison, WI, USA), respectively. The WT/MUT lncRNA RPSAP52 or WT/MUT STAT3 were co-transfected with miR-665 mimic/mimic-NC using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the guidance of manufacturer. Luciferase activity was detected via using Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA).
3
Evaluating SlMYC2 Transcription Factor
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The effector vector pBD-SlMYC2 was constructed using a seamless cloning kit (Beyotime, Shanghai, China). To mimic methionine sulfoxidation, Met542 in SlMYC2 was mutated to glutamine using a site-directed mutagenesis Kit (Beyotime, Shanghai, China), with the constructed vector pBD-SlMYC2 as a template, and transformed into DH5α chemically competent cell. Using the Agrobacterium strain GV3101 (pSoup-P19), the effector vector and reporter vector (pGreenII 0800-LUC-TATA) were co-infected into tobacco leaves. The values of LUC and REN were determined after 72 h using a dual-luciferase reporter gene assay kit (Beyotime, Shanghai, China).
4
Luciferase Reporter Assay for mRNA-lncRNA Interactions
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Luciferase reporter assays were carried out as previously described [48 (link)]. The CPCFC mRNA sequence including the 3′UTR and the lnc19419 sequence were each inserted into the vector pmirGLO (Promega). Mutated versions were prepared using a site-directed mutagenesis kit (Beyotime Biotechnology). The four plasmids were then used in separate transfection experiments, and were introduced into HEK-293 T cells along with synthetic miRNAs using transIt-LT1 transfection reagent (Mirus Bio, Madison, WI, USA). The relative luciferase activity was measured 24 h post-transfection with an Orion microplate luminometer (Berthold Technologies, Bad Wildbad, Germany) and the dual-glo luciferase assay system (Promega).
5
Site-Directed Plasmid Mutagenesis Protocol
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Point mutant plasmids were generated by using a site-directed mutagenesis kit (Beyotime, Shanghai, China). A total of 50 ng plasmid DNA was used for the reaction with the mutagenic oligonucleotides. All point mutations were verified via Sanger sequencing.
6
Validating miR-200b Regulation of ETS1
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To verify whether ETS1 was a direct target of miR-200b, the complete sequence of the ETS1 wild-type 3′-UTR (ETS1_UTR_WT) was amplified by PCR from human genomic DNA using the forward primer, 5′-GCTCTAGAGCTATCACTCTAGTTTTGAAGC-3′ and reverse primer, 5′-GCTCTAGAGCCTTTCATTGTGACAGAATCC-3′. Both primers contained the recognition sequence for the XbaI restriction enzyme at the 5′ ends. Subsequently, the sequence was cloned into the pGL3 (cat. no. 48743; Addgene) vector downstream of the luciferase open reading frame. Additionally, the site-directed mutagenesis for ETS1 3′-UTR was performed using a site-directed mutagenesis kit (Beyotime Institute of Biotechnology) to remove the miR-200b binding site.
Aliquots of 2×105 exponentially growing BEAS-2B cells were seeded into 24-well plates and co-transfected with the mimics (miR-200b) and the constructed pGL3 plasmids, as well as the Renilla luciferase plasmid (pRL, Addgene, cat. no. 27163, which was used as the internal control) at a ratio of 5:5:1 using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Lysates were collected 48 h after transfection. Firefly and Renilla luciferase activities were measured using a Firefly luciferase Reporter Gene Assay kit (Beyotime Institute of Biotechnology).
Aliquots of 2×105 exponentially growing BEAS-2B cells were seeded into 24-well plates and co-transfected with the mimics (miR-200b) and the constructed pGL3 plasmids, as well as the Renilla luciferase plasmid (pRL, Addgene, cat. no. 27163, which was used as the internal control) at a ratio of 5:5:1 using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Lysates were collected 48 h after transfection. Firefly and Renilla luciferase activities were measured using a Firefly luciferase Reporter Gene Assay kit (Beyotime Institute of Biotechnology).
7
miRNA Target Prediction and Validation
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Two miRNA target prediction software programs, miRanda (http://www.microrna.org/microrna/getDownloads.do ) [62 (link)] and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/welcome.html ) [63 (link)], were used with default parameters to predict miRNAs that potentially targeted the 3’ untranslated region (UTR) of the GSTu1 transcript. The luciferase reporter plasmid was constructed by inserting the wild-type or mutated target sequences of GSTu1 between the firefly luciferase ORF (Open Reading Frame) and SV40 poly (A) into the pmirGLO vector (Promega, Leiden, Netherlands). The mutated target sequences were synthesized using a Site-Directed Mutagenesis Kit (Beyotime, Nanjing, China) following the manufacturer’s instructions. For the luciferase assay, HEK293T cells were cultured in a 96-well plate and transfected with the reporter plasmids and miRNA agomir or NC agomir using a calcium phosphate cell transfection kit (Beyotime) following the manufacturer’s instructions. Each well contained 0.2 μg plasmid and 90 nM miRNA agomir. Luciferase assays were performed using the Dual-Glo Luciferase Assay System (Promega) at 24 h posttransfection. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was compared with activity in the control groups. For each transfection, luciferase activity was averaged from the results of six replicates.
8
Site-Directed Mutagenesis of Plasmids
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Point mutant plasmids were constructed using a site-directed mutagenesis kit (Beyotime, China). Five hundred nanograms plasmid DNA was used for the reaction with mutagenic oligonucleotides. All point mutations were verified via Sanger sequencing.
9
Cloning and Mutation of hsp90b2 3'-UTR
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3′-UTR segment of the hsp90b2 with the ssa-miR-301a-3p binding site was cloned into pmirGLO dual-luciferase vector (Promega, Madison, WI, USA), which was digested with restriction enzymes SacI and XhoI, and produced a wild-type structure pmirGL0-hsp90b2-wt-3′-UTR (hsp90b2 WT) by using ClonExpress® Entry One Step Cloning Kit (GenePharma, Shanghai, China). 3′-UTR mutation sequence of hsp90b2 pmirGLO-hsp90b2-mut-3′-UTR (hsp90b2 MUT) constructed by binding site (TTGCACTG) within the 3′-UTR of the hsp90b2 was changed using Site-Directed Mutagenesis Kit (Beyotime, ShangHai, China) and took a place of (AACGTGAC) (Fig. 1 ). At the same time, reverse complementary sequences of ssa-miR-301a-3p were cloned to pmirGLO dual-luciferase vector to construct positive control (ssa-miR-301a-3p PC), which was transferred to HEK 293T cells and was processed to siRNA (small interference RNA) segments of 21–23 bp in length by Dicer, and then siRNA recognized ssa-miR-301a-3p mimic with homologous sequences through base complementary ligation and mediated ssa-miR-301a-3p mimic degradation. All recombinant plasmids were tested for enzyme digestion and sequencing.
10
Cloning and Mutagenesis of DUSP6
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DUSP6 was amplified by PCR from HeLa cDNA and cloned into vector pCMV-HA between restriction enzymes Hind III and Kpn I under the control of a cytomegalovirus promoter, generating pCMV-HA-DUSP6 with HA-tag at the N-terminus. The dominant negative mutant pCMV-HA-DUSP6-DN (C293S) was generated using a Site Directed Mutagenesis Kit (Beyotime, Jiangsu, China). The primers for amplification of DUSP6 were 5′-AAGCCAACACCCTTCCAGTA-3′ (forward) and 5′-GCCCAGCTCTCTCTGACACA-3′ (reverse). The primers for mutation of DUSP6 were 5′-GTTCTGCTATGAGCTAGCTGGAGA GCCCTTGGTC-3′ (forward) and 5′-GACCAAGGGCTCTCCAGCTAGCTCATAGCAGAAC-3′ (reverse).
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