The GUSPlus reporter gene was PCR amplified from the pCAMBIA1305.2 and cloned into the pZP35S:RFP vector (containing a gentamycin resistance gene for transgenic plant selection) [88 (link)] to replace the RFP reporter gene with the help of BamHI and HindIII. The cDNA sequence of the cleistogamy-inducing PpJAZ1 gene (Accession #: EMJ03624) from peach was PCR amplified from the binary vector pGrII-35S-PpJAZ1-eGFP [70 (link)], fused in-frame with a red fluorescent protein pporRFP reporter gene [88 (link)–90 (link)], and cloned into the pCR8 vector (Thermo Fisher; Waltham, MA, USA). Following Sanger sequencing, the PpJAZ1-pporRFP was cloned into the binary vector pMDC32 (containing a hygromycin resistance gene for transgenic plant selection) under the control of the full-length CaMV 35S promoter using the LR reaction (Thermo Fisher; Waltham, MA, USA). The resulting destination plasmid was confirmed using Sanger sequencing, and named as the pMDC35S JAZ1-RFP (Supplementary Data Figure S1 ). The primers used in PCR amplification were listed in Supplementary Data Table S1 .
Pcr8 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PCR8 vector is a plasmid that can be used for cloning and sequencing of PCR products. It contains a multiple cloning site and antibiotic resistance markers for selection.
Automatically generated - may contain errors
Lab products found in correlation
T7 rna polymerase, by Roche (1 mentions)
Biotin rna labeling mix, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase free dnase 1, by Roche (1 mentions)
Advantage 2 polymerase, by Takara Bio (1 mentions)
Gateway technology, by Thermo Fisher Scientific (1 mentions)
Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
5 protocols using pcr8 vector
1
Cloning and Characterization of PpJAZ1-RFP Fusion Construct
2
Cloning AtPTP1 from Arabidopsis
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The coding sequence of AtPTP1 (At1g71860) was amplified by PCR from A. thaliana leaf cDNA using Phusion Taq Polymerase and then cloned into the PCR8 vector (Thermofisher). The coding sequence of AtPTP1 was then transferred into the pHGWA vector (Busso et al., 2005 (link)) by LR recombination, resulting in pHGWA-AtPTP1.
3
Biotin-labeled HEIH RNA Pulldown Assay
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Biotin-labeled HEIH was synthetized as the probe for RNA pull-down assay, and the binding of Ago2 was determined by western blot analysis and RT-qPCR. The DNA fragment of HEIH sequence was amplified with a primer containing a T7 promoter and cloned into a pCR8 vector (Invitrogen). Subsequently, the plasmid was treated with the restriction enzyme NotI. Biotin-labeled RNAs were reversely transcribed with Biotin RNA Labeling Mix (Roche Diagnostics, NJ, USA) together with T7 RNA polymerase (Roche, Basel, Switzerland). The product was purified with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) after treated with Rnase-free DNase I (Roche, IN, USA). After biotin-labeled HEIH was incubated with TE-1 and KYSE-30 cell lysate overnight, biotin-labeled HEIH was captured using streptavidin magnetic beads, and incubated for 1 h. Western blot analysis was adopted to detect biotin-labeled HEIH level, and RT-qPCR to determine miR-185 level in RNA precipitates.
4
Generation of Transgenic Tomato Plants with SlDOF10 Constructs
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To make the GUS reporter fusion, approximately 2.5-kb of the 5′ promoter region of the SlDOF10 gene was amplified using Advantage 2 Polymerase (Clontech) and oligonucleotides DOF10pro-For and DOF10pro-Rev, then cloned in the pCR8 vector (Invitrogen). Destination vector pKGWFS7,0 (VIB/Gent, Belgium) was used to generate the SlDOFpro::GUS construct. To make the 35S::SlDOF10 construct, SlDOF10 cDNA was amplified with oligos SlDOF10-ORF-for and SlDOF10-ORF-rev and inserted into pK2GW7 binary vector (VIB/Gent, Belgium) using Gateway technology (Invitrogen) that placed the cDNA under the cauliflower mosaic virus 35S promoter. The SlDOF10-RNAi construct was generated using a 428 bp fragment of SlDOF10 gene (positions 349–777 from the ATG codon), amplified using primers SlDOF10-RNAi-For and SlDOF10-RNAi-Rev and cloned into pK7GWIWG2(I) vector (VIB/Gent, Belgium). The fragment used in this construct is located outside of the conserved motifs of the protein (DOF domain and bipartite NLS signal). Primers are listed in Supplementary Table S1 .
These three binary vectors were then introduced into A. tumefaciens LBA4404 by electroporation. The cotyledon co-cultivation method (Ellul et al., 2003 (link)) was used to transform wild-type tomato plants (cv. Micro-Tom). The transgenic plants were screened on antibiotic plates and transformants were transferred to soil for propagation.
These three binary vectors were then introduced into A. tumefaciens LBA4404 by electroporation. The cotyledon co-cultivation method (Ellul et al., 2003 (link)) was used to transform wild-type tomato plants (cv. Micro-Tom). The transgenic plants were screened on antibiotic plates and transformants were transferred to soil for propagation.
5
Genetic Manipulation of OsCKX3 in Rice
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The target gRNA sequence of OsCKX3 was cloned into the CRISPR/Cas9 vector with the primers P3F and P3R to generate the osckx3 mutants. The primers P4F and P4R were used to identify the osckx3 mutants. OsCKX3 was amplified from cDNA using primers P5F and P5R. The coding sequence of OsCKX3 was cloned into the pCR8 vector (Invitrogen) using the sequence-and ligation-independent cloning method. The entry vector pCR8-OsCKX3 was cloned into the binary vector pMDC32 and pMDC43 to construct 35S::OsCKX3 and 35S::OsCKX3-GFP vectors by LR reaction using the Gateway LR Clonase II enzyme mix (11791-020, Invitrogen). In the same way, the OsCKX3 promoter sequence was amplified by primers P7F and P7R and cloned into the pMDC163 vector to generate the pOsCKX3::GUS construct. OsCKX3 full-length fragment was amplified using primers P6F and P6R from the pCR8-OsCKX3, and was cloned into pMAL-c2X vector to generate pMAL-OsCKX3 expression vector using Clonexpress II One Step Cloning Kit (Vazyme, Nanjing, China).
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