RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM-5′ RACE) was performed to map the cleavage sites of the target transcripts by using the GeneRacer kit (Invitrogen, USA). At 15 °C overnight, three replicates of l µg total RNA isolated from persimmon fruit samples were ligated with 5′ RACE RNA adaptors. Gene-specific primers (GSP) (Table S1 ) were designed to conduct 5′ RACE PCR. The PCR fragments were cloned into the pEASY-Blunt Simple vector (TransGen Biotech, Beijing, China) and subjected to sequencing.
Peasy blunt simple vector
Manufactured by Transgene
Sourced in China
PEASY-Blunt Simple vector is a basic cloning vector designed for the insertion and expression of DNA sequences. It provides a simple and efficient method for the cloning of DNA fragments with blunt ends. The vector contains a multiple cloning site, an antibiotic resistance marker, and a bacterial origin of replication.
Automatically generated - may contain errors
Lab products found in correlation
Generacer kit, by Thermo Fisher Scientific (2 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Firstchoice rlm race kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Race polymerase chain reaction, by Sangon (2 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Pspax2, by Addgene (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Smarter race 5 3 kit, by Takara Bio (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Primerscript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Pcold 2 vector, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Gel extraction kit, by Vazyme (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
21 protocols using peasy blunt simple vector
1
Mapping Transcripts Cleavage Sites
2
CRISPR/Cas9-Mediated Gene Editing in Mice
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pSpCas9(BB)‐2A‐GFP (pX458) was a gift from Feng Zhang (Addgene plasmid # 48138). The protospacer sequence targeting the Y381D mouse locus was synthesized from Genewiz and inserted into pX458 through BbsI. The HDR donor vector was constructed by insertion of the donor sequence into the pEASY‐Blunt Simple vector (TransGen Biotech). ssODNs (120 nt; Appendix Table S2 ) carrying the D381Y were synthesized from Genewiz. Hydrodynamic injection was performed as described previously (Liu et al, 1999 ) with minor modifications. Briefly, all dosages of plasmid solution were diluted into 1 ml (final volume) 0.9% NaCl. The tail vein injection was performed within 5–7s for maximum liver absorption. For the plasmid donor group, mice were injected with both pX458 (120 μg) and pEASY‐HDR donor (120 μg); for the ssODN donor group, mice were injected with pX458 (120 μg) and ssODN‐HDR donor (120 μg). The control group received 1 ml 0.9% NaCl only.
3
Isolation and Characterization of BcCUC2 Gene
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Total RNA was extracted from leaves using RNAprep pure Plant Kit (TIANGEN, Beijing, China), and the first strands of cDNA were synthesized via reverse transcription using PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The coding sequence (CDS) of BcCUC2 was amplified by gene-specific primers (see Table A1 ) based on the sequence of Bra022685 using homology cloning referred to in our previous report [25 (link)]. The resulting fragment was cloned into pEASYBlunt Simple Vector (Transgene, Beijing, China) and sequenced by Genscript Company (Nanjing, China). The physicochemical characteristics of BcCUC2 were predicted using Expasy proteomics server (https://web.expasy.org/protparam/ , accessed on 10 July 2020). The secondary structure of BcCUC2 was predicted using PSIPRED 4.0 (http://bioinf.cs.ucl.ac.uk/psipred/ , accessed on 10 July 2020).
4
Mapping Transcript Cleavage Sites via RLM-5' RACE
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RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM-5′ RACE) was conducted with a GeneRacer kit (Invitrogen, Carlsbad, CA, United States) to map the cleavage sites of the target transcripts. Three replicates of l μg of total RNA isolated from pooled samples of persimmon fruits were subjected to the ligation of 5′ RACE RNA adaptors at 15°C overnight. Gene-specific primers (Supplementary Table S1 ) were designed to conduct 5′ RACE PCR. The PCR products were cloned into the pEASY-Blunt Simple vector (TransGen Biotech, China) for sequencing.
5
Lentiviral Vector Production Protocol
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To generate lentiviral vectors, EGFP-TTALE cassettes were inserted into the modified pEASY-Blunt Simple vector (TransGen Biotech) using SacI and AscI, and finally cloned into the pLE4 lentiviral vector (a kind gift from Dr Tomoaki Hishida) using BamHI and MluI. To package lentivirus constructs, HEK293T cells were transfected with lentiviral vectors together with the packaging plasmids pMD2.G (Addgene, 12260) and psPAX2 (Addgene, 12259). Supernatants containing viruses were harvested at 48 and 72 h after transfection, filtered with a 0.45-μm PVDF membrane (Millipore), concentrated by ultracentrifugation at 19 400× g for 2.5 h, and assessed for viral titers.
6
Porcine Subcutaneous Fat Transcriptome Profiling
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5′-RACE and 3′-RACE experiments were performed using a FirstChoice RLM-RACE kit (Thermo, Otsu, Japan) and a SMARTer RACE 5′/3′ kit (TakaRa, Otsu, Japan) according to the manufacturer’s protocols, respectively. Primer sequences of the RACE experiment are shown in Table S1 . Total RNA isolated from porcine subcutaneous fat was used as templates in nested PCR reactions. The 5′- and 3′-RACE products were gel-purified using an Axygen AxyPrep DNA gel extraction kit (Axygen, Guangzhou, China) and cloned into the pEASY-blunt simple vector (Transgen, Beijing, China) following the manufacturer’s instructions, which were subsequently sequenced and analyzed.
7
Inverse-nested PCR for T-DNA flanking
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The T-DNA flanking region was isolated by inverse-nested PCR using the inverse primer designed from the 35S promoter and hygromycin sequences as described in Uchiyama and Watanabe55 (link). The PCR products were purified, cloned into the pEASY- Blunt Simple vector (Transgen Biotech, Beijing, China) and sequenced.
8
Cloning and Mutagenesis of Plant Genes
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The full open reading frames or fragments of CDC48A, HSP70, HSP90, TOC33, TIC195, WHY2 (Nuc), WHY2 (Mito), WHY2 (Chl), UPL5 were amplified by PCR from Col-0 cDNA. Appropriate restriction sites were included within the primers for follow-up cloning after subcloning the DNA fragments into the pEASY-Blunt Simple vector (Trans-Gen). All clones were validated by DNA sequencing. The following destination vectors were used: pGBK-T7 (Clontech), pGAD-T7 (Clontech), pBiFC-nYFP/cYFP (Miao et al., 2007), pRTDVcVN/pRTDVcVC.36 (link) Specifically, for the construction of WHY2 (K45R), WHY2 (K227R), UPL5 (C389S), The UPL5 mutant gene (C839S) was generated using a QuikChange site-directed mutagenesis kit (Agilent). Primers for all constructs generated in this study are listed in Table S1 .
9
Cloning and Expression of ADH Genes
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Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed (RT) using a the PrimerScript First Strand cDNA Synthesis Kit (TaKaRa) with random primers and oligo(dT) at the same time. BLAST searches against unigenes of cluster 3 and cluster 6 in A. euchroma transcriptome database resulted in two candidate genes, unigene18140 (AeHGO) and unigene32185, with sequence similarity to alcohol dehydrogenases (ADHs). Their full-length clones were acquired by PCR using the gene-specific primer pairs AeHGO-F1 (5′-TATGGAGCTCGGTACC ATGGGAAATTCAGCAGAAC-3′) and AeHGO-R1 (5′- CGACAAGCTTGAATTC TTAGGCAGTTTTAAGAGTATTGC-3′), 32185-F1 (5′- TATGGAGCTCGGTACC ATGTCCAACACTGCTGG-3′) and 32185-R1 (5′- CGACAAGCTTGAATTC TTAACCTTCCATGTTGATAATGC-3′). In these primers the extensions homologous to vector ends for subcloning were underlined. The PCR products were cloned into pEASY-Blunt Simple vector (TransGen) for sequencing and then subcloned into pCold II vector (Takara) using In-Fusion HD Cloning Kit (Takara), which resulted in the expression construct pCold II-AeHGO and pCold II-unigene32185.
10
Cloning and Sequencing of CsGLK Genes
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Total RNA was extracted from the tea plants using an RNA Extraction Kit (Biofit, Beijing, China) according to the manufacturer’s instructions. Reverse transcription reactions were performed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The gene was amplified with PrimeSTAR® Max DNA Polymerase (Takara, Dalian, China) using cDNA. The PCR products were purified with a Gel Extraction Kit (Vazyme, Nanjing, China), ligated into a pEasy-Blunt Simple vector (TransGen Biotech, Beijing, China), and subsequently transformed into Trans-T1-competent cells for sequencing. The primers of CsGLK1 (accession number MZ093621) and CsGLK2 (MZ093620) are listed in Supplementary Data Table S3 .
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