To generate the cMYC-OsTIR1 construct, the coding region of OsTIR1 was amplified by PCR, cloned into the pEASY–Blunt vector (Transgene, China), and subcloned into the pE3n vector71 (link). The cMYC-OsTIR1 fragment was then inserted into the pCAMBIA2300 vector downstream of the maize (Zea mays) Ubiquitin promoter after KpnI and BamHI digestion. To generate the cMYC-OsIAA6 construct, the coding region of OsIAA6 was amplified by PCR, cloned into the pEASY-Blunt vector (Transgene, China), and subcloned into the pE3n vector. The cMYC-OsIAA6 fragment was then inserted into the pCAMBIA2300 vector downstream of the maize (Zea mays) Ubiquitin promoter after BamHI and NotI digestion. All of the primers used to generate the aforementioned constructs are listed in Supplemental Table S1 , and all of the constructs were confirmed by sequencing. The constructed vectors were transformed by Agrobacterium tumefaciens strain EHA105. Wild type ZH11 calli were used as the recipients for Agrobacterium-mediated transformation as described72 .
Peasy blunt vector
Manufactured by Transgene
Sourced in China, United States
PEASY-Blunt vector is a plasmid-based cloning vector designed for blunt-end cloning. The vector provides a simple and efficient method for inserting DNA fragments with blunt ends into the plasmid. It can be used in various molecular biology applications that require the cloning of blunt-ended DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Axyprep dna gel extraction kit, by Corning (5 mentions)
Kod plus dna polymerase, by Toyobo (4 mentions)
Sanger sequencing, by Sangon (3 mentions)
Fastpfu dna polymerase, by Transgene (3 mentions)
Saccharomyces cerevisiae strain ah109, by Takara Bio (3 mentions)
Smarter race 5 3 kit, by Takara Bio (3 mentions)
Smarter race cdna amplification kit, by Takara Bio (2 mentions)
Transstart fastpfu dna polymerase, by Transgene (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Primestar hs dna polymerase, by Takara Bio (2 mentions)
Easypfu dna polymerase, by Transgene (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Tcs sp8, by Leica (2 mentions)
M13f r primers, by Sangon (2 mentions)
Peasy blunt cloning kit, by Transgene (2 mentions)
Sanprep column dna gel extraction kit, by Sangon (2 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (2 mentions)
Genome extraction kit, by CWBIO (2 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (2 mentions)
Primestar max premix, by Takara Bio (2 mentions)
96 protocols using peasy blunt vector
1
Generating cMYC-OsTIR1 and cMYC-OsIAA6 Constructs
2
Isolation of AfAP2-1 Gene from A. fasciata
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To isolate the AfAP2-1 gene, total RNA was extracted from the central leaves of A. fasciata using Hexadecyl trimethyl ammonium bromide (CTAB) methods [39 (link)]. Based on the transcriptome data of A. fasciata, 5′ and 3′ RACE was performed using a SMARTer™ RACE cDNA Amplification Kit (Clontech, Tokyo, Japan) according to the manufacturer’s instructions. The primers used here are listed in Table S1 . The specific 5′ and 3′ fragments were cloned into pEASY-blunt vectors (Transgene, Beijing, China) and sequenced by Thermo Fisher Scientific (Guangzhou, China).
3
Isolation and Sequencing of AfSPL14
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Total RNA was extracted from the central leaves of A. fasciata using the hexadecyl trimethyl ammonium bromide (CTAB) method [33 (link)], and then used for the RACE at the 5′ and 3′ ends according to the manufacturer’s instructions for the SMARTer™ RACE cDNA Amplification Kit (Clontech, Tokyo, Japan). The specific 5′ and 3′ fragments were cloned into pEASY-blunt vectors (Transgen, Beijing, China), and then sequenced by Thermo Fisher Scientific (Guangzhou, China). The gene accession number of AfSPL14 is MF114304. The primers used here are listed in Table S3 online.
4
Cloning and Plasmid Modification of OmpK35 and OmpK36
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The coding sequences of wild type ompk35 and ompk36 genes from reference isolate K. pneumoniae ATCC 13883 were amplified using primer pairs bluntOmpk35 (forward, 5′-AATGATGAAGCGCAATATTCT-3′; reverse, 5′- CGAAGGGGTGTACTGCAGATTA-3′) and bluntOmpk36 (forward, 5′- CATGAAAGTTAAAGTACTGTC-3′; reverse, 5′- TTATGCAGCTTGCAACTTAGAA-3′), respectively, and subsequently cloned into pEASY-Blunt vectors (Transgen Biotech Co., China). After nucleotide sequence verification, the kanamycin resistance gene of recombinant plasmids was replaced by the apramycin resistance gene using PCR. As a control, the kanamycin resistance gene in an empty vector was also replaced by the apramycin resistance gene. The recombinant plasmids were selected on Luria-Bertani (LB) agar containing apramycin at a concentration of 50 μg/ml.
5
Cloning and Sequencing of PCR Products
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The PCR products were determined by agarose gel electrophoresis (150 V, 30 min). The concentration of One microliter of pEASY-Blunt vectors (TransGen Biotech, Beijing, China) and 2 μL of DNA were ligated at 23-28 °C for 10 min. Fifty microliters of Trans-T1 competent cells were added to the mixture, which were chilled on ice for 20 min, heated to 42 °C for 45 s, and chilled on ice for 2 min. The mixtures were transferred to 5-mL centrifuge tubes that contained 800 μL of Luria-Bertani (LB) medium, and the tubes were kept at 37 °C rotating at <200 rpm for 1 h. Two hundred microliters of the bacterial solution was added to solid LB media that contained ampicillin (Amp), isopropyl β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-4chloro-3-indolyl-D-galactoside (X-gal); the media were cultured at 37 °C overnight. Eight promising colonies were picked and cultured in 1 mL of liquid LB medium supplemented with 1 μL of Amp at 37 °C and 200 rpm for 5 h. After PCR detection, 5 positive clones were selected in each sample for sequencing by Sanger sequencing (Sangon Biotech, Shanghai, China).
6
PEDV S Gene Sequence Analysis
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The positive samples whose CT values are less than 30 were selected for S1 region sequencing. To obtain the S1 region sequences, PCR was conducted with the specific primer pair of PEDV-S1-F and PEDV-S1-R listed in Supplementary Table S1 . To amplify the S gene of the NH-TA2020 strain, four pairs of primers were used for PCR, including PEDV-S-1-F&PEDV-S-1-R, PEDV-S-2-F&PEDV-S-2-R, PEDV-S-3-F&PEDV-S-3-R, PEDV-S-4-F&PEDV-S-4-R (Supplementary Table S1 ). PCR products were analyzed by electrophoresis in 1.0% agarose gels. The fragments were cloned into pEASY-blunt vectors (TransGen Biotech, Beijing), and the positive clones were sequenced at Sangon Biotech (Qingdao, China). The phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA v.7.1.0 (44) software with the default settings (1000 bootstrap replicates) based on the PEDV S gene sequences sequenced in this study and reference sequences collected in GenBank. Initial tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The resulting tree was visualized using iTOL v.6 (Interactive Tree of Life, http://itol.embl.de/ ).
7
Cloning and Sequencing of PCR Products
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The PCR products were determined by agarose gel electrophoresis (150 V, 30 min). The concentration of One microliter of pEASY-Blunt vectors (TransGen Biotech, Beijing, China) and 2 μL of DNA were ligated at 23-28 °C for 10 min. Fifty microliters of Trans-T1 competent cells were added to the mixture, which were chilled on ice for 20 min, heated to 42 °C for 45 s, and chilled on ice for 2 min. The mixtures were transferred to 5-mL centrifuge tubes that contained 800 μL of Luria-Bertani (LB) medium, and the tubes were kept at 37 °C rotating at <200 rpm for 1 h. Two hundred microliters of the bacterial solution was added to solid LB media that contained ampicillin (Amp), isopropyl β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-4chloro-3-indolyl-D-galactoside (X-gal); the media were cultured at 37 °C overnight. Eight promising colonies were picked and cultured in 1 mL of liquid LB medium supplemented with 1 μL of Amp at 37 °C and 200 rpm for 5 h. After PCR detection, 5 positive clones were selected in each sample for sequencing by Sanger sequencing (Sangon Biotech, Shanghai, China).
8
Cloning and Sequencing of ZmYSL Genes
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The cDNA sequences of the ZmYSL genes with complete ORFs were retrieved from the MaizeSequence database (http://www.maizesequence.org/ ). The ORFs of the ZmYSL genes were amplified using the primers listed in Table S8 . The cDNA was synthesized following the manufacturer’s instructions (TransGen Biotech Co., Ltd., China), and the high-fidelity DNA polymerase KOD FX Neo (TOYOBO) was used for amplifying the target genes. PCR amplification was conducted using a DNA amplification machine (Eppendorf). The PCR products were separated on a 1.0% agarose gel and purified with the FastPure Gel DNA Extraction Mini Kit (Vazyme) following the manufacturer’s instructions. The purified product was cloned into the pEASY-Blunt vector (TransGen Biotech Co., Ltd., China) and sequenced (Openlab, China).
9
Cloning and Characterizing Insect Gustatory Receptors
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We referred to published transcriptome data [19 (link)] to screen for Grs expressed in larval mouthparts. As these predicted BmGrs sequences were incomplete or inaccurate, specific primers were designed to obtain full-length coding sequences. PCR was conducted using Platinum SuperFi II Green PCR Master Mix (Invitrogen, Carlsbad, CA, USA) as follows: 98°C for 30 s, followed by 35–40 cycles of 98°C for 10 s, 60°C for 10 s and 72°C for 1 min, and a final extension at 72°C for 5 min. Amplified BmGrs were then cloned into a pEASY-Blunt vector (TransGen Biotech, Beijing, China) and sequenced. Grs that terminated early or were un-cloned were removed and the rest were used as candidate Grs for follow-up experiments (electronic supplementary material, table S2).
To confirm BmGr63 localization in human embryonic kidney 293T (HEK293T) cells, an mScarlet tag with red fluorescence was fused with BmGr63 into the pcDNA3.1 vector. The full-length BmGr63 coding sequence was inserted between BglII and BcuI sites in the pT7Ts expression vector and used for cRNA synthesis.
To confirm BmGr63 localization in human embryonic kidney 293T (HEK293T) cells, an mScarlet tag with red fluorescence was fused with BmGr63 into the pcDNA3.1 vector. The full-length BmGr63 coding sequence was inserted between BglII and BcuI sites in the pT7Ts expression vector and used for cRNA synthesis.
10
Isolation and Identification of T-DNA Insertion Sites
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TAIL‐PCR (Liu et al., 1995 ) was used to isolate regions flanking the T‐DNA insertion with Genome Walking Kit (TaKaRa, Tokyo, Japan). PCR‐amplified fragments were cloned into the pEASY‐Blunt vector (TransGen Biotech, China) and then sequenced (Biomed Biotech, Beijing, China). For Southern blotting, about 10 μg genomic DNA from wild‐type and spl35 mutant were digested with HindIII overnight, and digested products were separated in 1% agarose gels and then transferred onto a Hybond‐N+ membrane (Amersham Pharmacia, Biotech, Buckinghamshire, UK). A 481‐bp fragment of the hygromycin phosphotransferase gene amplified from the pCUbi1390 vector by the primer pair Hyr‐F/R was DIG‐labelled and used as a hybridization probe. Southern blotting was performed with DIG high prime DNA labelling and a detection starter kit II according to the manufacturer's instructions (Roche, Mannheim, Germany). Primers used for TAIL‐PCR and Southern blotting are listed in Table S1 .
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