Structured Review

TaKaRa in fusion hd cloning kit
In Fusion Hd Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/in fusion hd cloning kit/product/TaKaRa
Average 99 stars, based on 1616 article reviews
Price from $9.99 to $1999.99
in fusion hd cloning kit - by Bioz Stars, 2020-04
99/100 stars

Images

Related Articles

Clone Assay:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. The pC003-LshC2C2 locus included in the plasmid pACYC184 for spacer cloning was a gift from Dr. Feng Zhang (Addgene plasmid #79152 ; RRID:Addgene_79152 ) ( ).

Article Title: APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression
Article Snippet: .. All the amplicons were cloned into the lentiviral plasmid pCSII–CMV–MCS (RIKEN, RDB04377) by using the In-Fusion HD Cloning Kit (TaKaRa), to produce the pCSII–CMV:A3B–3×FLAG–IRES–EGFP donor DNA plasmid ( ). .. Validation of sgRNA targeting efficiency 293T cells were transfected with pSpCas9(BB)–2A–Puro:sgRNA #4 (0.5 μg) using the FuGENE HD Transfection Reagent (Promega).

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: .. C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan). .. Protein expression in HEK cells PACs were expressed in HEK293 cells that stably expressed a GloSensor-22F cAMP, as previously described .

Article Title: Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells
Article Snippet: .. The Tet-On 3G transactivator of the pTetOne vector (Takara) was PCR-amplified and cloned into the multicloning site of the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara). .. The resulting vector was designated as pCS-CA-Tet3G.

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The insertion sequence was prepared by PCR using the amplified LC sequence as a template and the following primer sets: Forward: CTC GAG CTC AAG CTT GCC GCC ACC ATG GTG CTG, Reverse: CTC GCC CTT GCT CAC TGA TCC TCC TCC TCC TGA.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: .. In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product. .. The plasmid pEGFP-N1 was used as control plasmid.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: .. The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. The recombinant plasmids, pET30a-CHH1 and pET30a-CHH2, were transformed into E. coli BL21 (DE3) competent cell for sequencing.

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: .. Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech). .. To generate the enCRISPRi-LK sgRNA vector, the KRAB sequence was PCR amplified from the pLV hU6-sgRNA hUbC-dCas9-KRAB-T2A-Puro vector (Addgene, plasmid #71236) and cloned into the enCRISPRa sgRNA vector to replace VP64.

Article Title: Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate
Article Snippet: .. The fragments P8-F and efALS -F were inserted the vector pLC0 -L simultaneously by using the In-Fusion® HD Cloning Kit ( https://www.takarabio.com ) to generate the plasmid pLC01. .. The plasmids pLC02 and pLC17 were constructed in the same way but with the fragments wxALS -F and llALS -F , respectively.

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: .. To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan). .. The individual luciferase gene was PCR amplified and cloned into pCAMBIA-N-GUS at NcoI/SpeI and PmlI sites by In-Fusion cloning.

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. 3′ End of RLD1 CDS (258 bp), corresponding to the BRX domain, was fused with DNA fragments containing mCherry gene and NOS terminator (mCherry-NOSt ) on the pUC19 vector under the cauliflower mosaic virus 35S promoter (35S:BRXd-mCherry ). mCherry gene without stop codon and LTI6b CDS were amplified by PCR and inserted into the multi-cloning site on the pUC19 vector using In-Fusion HD Cloning Kit (35S:mCherry-LTI6b ).

Article Title: Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains
Article Snippet: .. The CoreTetCT gene was cloned in plasmid pGEX-6P-1 using an In-Fusion HD Cloning Kit (Clontech, Mountain View, CA). ..

Amplification:

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The insertion sequence was prepared by PCR using the amplified LC sequence as a template and the following primer sets: Forward: CTC GAG CTC AAG CTT GCC GCC ACC ATG GTG CTG, Reverse: CTC GCC CTT GCT CAC TGA TCC TCC TCC TCC TGA.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: The nucleotide sequences which encoded the predicted signal peptide and TM domains were amplified using primers LvG C-EGFP-F/R. .. In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product.

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: To generate the enCRISPRa sgRNA vector, the MCP-VP64-IRES-mCherry cassette was PCR amplified from the pJZC34 vector (Addgene, plasmid #62331) and cloned into Bsr GI/Eco RI digested lenti-sgRNA (MS2)-zeo backbone (Addgene, plasmid # 61427). .. Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech).

Article Title: Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate
Article Snippet: The DNA fragment llALS -F was amplified from genomic DNA of Lactococcus lactis MG1363 using primers ALS -Ll -CF1 and ALS -Ll -CR1 (see Additional file : Table S1). .. The fragments P8-F and efALS -F were inserted the vector pLC0 -L simultaneously by using the In-Fusion® HD Cloning Kit ( https://www.takarabio.com ) to generate the plasmid pLC01.

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: .. To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan). .. The individual luciferase gene was PCR amplified and cloned into pCAMBIA-N-GUS at NcoI/SpeI and PmlI sites by In-Fusion cloning.

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. 3′ End of RLD1 CDS (258 bp), corresponding to the BRX domain, was fused with DNA fragments containing mCherry gene and NOS terminator (mCherry-NOSt ) on the pUC19 vector under the cauliflower mosaic virus 35S promoter (35S:BRXd-mCherry ). mCherry gene without stop codon and LTI6b CDS were amplified by PCR and inserted into the multi-cloning site on the pUC19 vector using In-Fusion HD Cloning Kit (35S:mCherry-LTI6b ).

Filtration:

Article Title: Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains
Article Snippet: The CoreTetCT gene was cloned in plasmid pGEX-6P-1 using an In-Fusion HD Cloning Kit (Clontech, Mountain View, CA). .. We confirmed the dimerization of the L334A mutant and the tetramerization of FL-p53 using a gel filtration column (Superdex 200; GE Healthcare, Tokyo, Japan).

Stable Transfection:

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: With binary vectors carrying these constructions, plants were stably transformed using standard protocol for Agrobacterium (strain GV3101)-mediated transformation . .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech).

Synthesized:

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan). .. The cDNAs of nnH3H and nnLuz were codon optimized for A . thaliana and synthesized (FASMAC, Kanagawa, Japan).

Construct:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: Plasmid Construction To examine the collateral growth inhibition activities of various Cas13a loci of Leptotrichia species, two sets of vectors were constructed. .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a.

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: Paragraph title: Constructs and mutants ... C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan).

Article Title: Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells
Article Snippet: Vectors for Dox-inducible p57 KIP2 expression were constructed as follows. .. The Tet-On 3G transactivator of the pTetOne vector (Takara) was PCR-amplified and cloned into the multicloning site of the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara).

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: The expression vector of IgG1 whose light chains (LC) are fused with Citrine at the C-terminus via a (G4 S)2 flexible linker (Fig. a, Haas et al. ) was constructed as follows. .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan).

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: Subcellular Localization of LvGC Protein in Mammalian 293T Cells To study the subcellular localization of LvGC, a plasmid containing the predicted signal peptide and transmembrane motif (TM) of LvGC was constructed, designed as pEGFP-LvGC. .. In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product.

Article Title: Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate
Article Snippet: Construction of the plasmids pLC0, pLC01, pLC02, and pLC17 PCR products pLC0 -L amplified from the plasmid pTD6 with the primers pTD -CF1 and pTD -CR1 (see Additional file : Table S1) were circularized using T4 DNA ligase after phosphorylation to construct the plasmid pLC0. .. The fragments P8-F and efALS -F were inserted the vector pLC0 -L simultaneously by using the In-Fusion® HD Cloning Kit ( https://www.takarabio.com ) to generate the plasmid pLC01.

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: .. To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan). .. The individual luciferase gene was PCR amplified and cloned into pCAMBIA-N-GUS at NcoI/SpeI and PmlI sites by In-Fusion cloning.

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. 3′ End of RLD1 CDS (258 bp), corresponding to the BRX domain, was fused with DNA fragments containing mCherry gene and NOS terminator (mCherry-NOSt ) on the pUC19 vector under the cauliflower mosaic virus 35S promoter (35S:BRXd-mCherry ). mCherry gene without stop codon and LTI6b CDS were amplified by PCR and inserted into the multi-cloning site on the pUC19 vector using In-Fusion HD Cloning Kit (35S:mCherry-LTI6b ).

Article Title: Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains
Article Snippet: The S392E mutant was constructed based on FL-p53 . .. The CoreTetCT gene was cloned in plasmid pGEX-6P-1 using an In-Fusion HD Cloning Kit (Clontech, Mountain View, CA).

Luciferase:

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: The GloSensor-22F cAMP gene (Promega Corporation, Fitchburg, WI, USA), a luciferase-based cAMP reporter, was cloned into a pEBMulti-Neo vector (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for monitoring the cAMP level in HEK293 cells. .. C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan).

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: Plasmid construction We used pCAMBIA-N-GUS as the backbone vector to express luciferase genes under the control of a 35S promoter. .. To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan).

Expressing:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: One set was for the expression of CRISPR-Cas13a and the other for the generation of their target RNA. .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a.

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: Constructs and mutants PAC genes with Rudolph-RFP and 2A-peptides at the N-terminus (RFP-2A-PACs) were cloned into pEBMulti-Hyg vectors (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for expression in HEK293 cells. .. C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan).

Article Title: Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells
Article Snippet: Vectors for Dox-inducible p57 KIP2 expression were constructed as follows. .. The Tet-On 3G transactivator of the pTetOne vector (Takara) was PCR-amplified and cloned into the multicloning site of the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara).

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: Paragraph title: Construction of the IgG-Citrine expression vector ... The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan).

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: Paragraph title: Expression and Purification of Recombinant Protein ... The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States).

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: To generate the inducible dCas9-LSD1 expression vector, LSD1 open-reading frame (ORF) was amplified and cloned into the pHR-TRE3G-KRAB-dCas9-P2A-mCherry to replace the KRAB domain. .. Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech).

BIA-KA:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. These recombinant proteins were examined by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and quantified to concentration of 500 ng μL–1 by BCA Protein Assay Kit (Tiangen, Beijing) for use.

Modification:

Article Title: APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression
Article Snippet: For the construction of the donor DNA vector , the right homology arm, the modified cassette including the 3×FLAG–IRES–EGFP gene and the left homology arm were PCR-amplified using KOD FX Neo (ToYoBo). .. All the amplicons were cloned into the lentiviral plasmid pCSII–CMV–MCS (RIKEN, RDB04377) by using the In-Fusion HD Cloning Kit (TaKaRa), to produce the pCSII–CMV:A3B–3×FLAG–IRES–EGFP donor DNA plasmid ( ).

Transformation Assay:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. The recombinant plasmids, pET30a-CHH1 and pET30a-CHH2, were transformed into E. coli BL21 (DE3) competent cell for sequencing.

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: With binary vectors carrying these constructions, plants were stably transformed using standard protocol for Agrobacterium (strain GV3101)-mediated transformation . .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech).

Over Expression:

Article Title: Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells
Article Snippet: Paragraph title: Overexpression of p57 KIP2 . ... The Tet-On 3G transactivator of the pTetOne vector (Takara) was PCR-amplified and cloned into the multicloning site of the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara).

Transfection:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product. .. Two micrograms of the plasmids pEGFP-LvGC and pEGFP-N1 were transfected into the mammalian 293T cells with Lipofectamine 3000 Reagent (Thermo Fisher Scientific, United States) following the manufacture’s instruction, respectively.

Inverse PCR:

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The Citrine-N1 vector was linearized by inverse PCR using the following primer sets to remove multi-cloning site sequences and the first ATG start codon of the Citrine gene: Forward: GTG AGC AAG GGC GAG GAG CT, Reverse: AAG CTT GAG CTC GAG ATC TG.

Generated:

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: .. C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan). .. Protein expression in HEK cells PACs were expressed in HEK293 cells that stably expressed a GloSensor-22F cAMP, as previously described .

Article Title: Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains
Article Snippet: The CoreTetCT gene was cloned in plasmid pGEX-6P-1 using an In-Fusion HD Cloning Kit (Clontech, Mountain View, CA). .. The dimer mutant genes in pGEX-6P-1 were generated using a PrimeSTAR Mutagenesis Basal Kit (TaKaRa, Shiga, Japan).

Inhibition:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: Plasmid Construction To examine the collateral growth inhibition activities of various Cas13a loci of Leptotrichia species, two sets of vectors were constructed. .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a.

Sequencing:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. Then the target sequence of the native Cas13a was inserted into just behind the RFP coding sequence.

Article Title: APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression
Article Snippet: sgRNA design and construction of A3B reporter donor DNA To design the single-guide RNA (sgRNA), the mRNA sequence of APOBEC3B (APOBEC3B Homo sapiens chromosome 22, GRCh38 Primary Assembly mRNA variant1, ) was imported into CRISPRdirect [ ]. .. All the amplicons were cloned into the lentiviral plasmid pCSII–CMV–MCS (RIKEN, RDB04377) by using the In-Fusion HD Cloning Kit (TaKaRa), to produce the pCSII–CMV:A3B–3×FLAG–IRES–EGFP donor DNA plasmid ( ).

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The insertion sequence was prepared by PCR using the amplified LC sequence as a template and the following primer sets: Forward: CTC GAG CTC AAG CTT GCC GCC ACC ATG GTG CTG, Reverse: CTC GCC CTT GCT CAC TGA TCC TCC TCC TCC TGA.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: Primers LvCHH1-rpF/rpR, LvCHH2-rpF/rpR were designed with 15 bp extension homologous of linearized pET30a vector (Novogen, France) ends at both ends to amply the ORF of LvCHH1 and LvCHH2 excluding sequence encoding signal peptide, respectively. .. The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States).

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech). .. To generate the enCRISPRi-LK sgRNA vector, the KRAB sequence was PCR amplified from the pLV hU6-sgRNA hUbC-dCas9-KRAB-T2A-Puro vector (Addgene, plasmid #71236) and cloned into the enCRISPRa sgRNA vector to replace VP64.

Recombinant:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: Paragraph title: Expression and Purification of Recombinant Protein ... The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States).

Mutagenesis:

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. In addition, we introduced mutations one by one at F1052 and W1066 of RLD1 on the RLD1p:RLD1-GFP vector by PCR amplification using primers containing respective mutation and then mutated RLD1 CDS was replaced with wild-type CDS on 35S:RLD1-GFP vector [35S:RLD1(F1052A&W1066A)-GFP , 35S:RLD1(F1052R&W1066R)-GFP ].

Article Title: Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains
Article Snippet: We prepared the CoreTetCT mutant, corresponding to residues 94–393. .. The CoreTetCT gene was cloned in plasmid pGEX-6P-1 using an In-Fusion HD Cloning Kit (Clontech, Mountain View, CA).

Isolation:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. The pC003-LshC2C2 locus included in the plasmid pACYC184 for spacer cloning was a gift from Dr. Feng Zhang (Addgene plasmid #79152 ; RRID:Addgene_79152 ) ( ).

Polymerase Chain Reaction:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. The pC003-LshC2C2 locus included in the plasmid pACYC184 for spacer cloning was a gift from Dr. Feng Zhang (Addgene plasmid #79152 ; RRID:Addgene_79152 ) ( ).

Article Title: APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression
Article Snippet: Each PCR primer pair contained around 15 bp overlaps. .. All the amplicons were cloned into the lentiviral plasmid pCSII–CMV–MCS (RIKEN, RDB04377) by using the In-Fusion HD Cloning Kit (TaKaRa), to produce the pCSII–CMV:A3B–3×FLAG–IRES–EGFP donor DNA plasmid ( ).

Article Title: Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells
Article Snippet: .. The Tet-On 3G transactivator of the pTetOne vector (Takara) was PCR-amplified and cloned into the multicloning site of the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara). .. The resulting vector was designated as pCS-CA-Tet3G.

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The insertion sequence was prepared by PCR using the amplified LC sequence as a template and the following primer sets: Forward: CTC GAG CTC AAG CTT GCC GCC ACC ATG GTG CTG, Reverse: CTC GCC CTT GCT CAC TGA TCC TCC TCC TCC TGA.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: .. In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product. .. The plasmid pEGFP-N1 was used as control plasmid.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: .. The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. The recombinant plasmids, pET30a-CHH1 and pET30a-CHH2, were transformed into E. coli BL21 (DE3) competent cell for sequencing.

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: To generate the enCRISPRa sgRNA vector, the MCP-VP64-IRES-mCherry cassette was PCR amplified from the pJZC34 vector (Addgene, plasmid #62331) and cloned into Bsr GI/Eco RI digested lenti-sgRNA (MS2)-zeo backbone (Addgene, plasmid # 61427). .. Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech).

Article Title: Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate
Article Snippet: The DNA fragment wxALS -F was obtained by PCR, using the primer pair WX02 -ALS -F/WX02 -ALS -R (see Additional file : Table S1) and synthetic DNA as template (see Additional file ). .. The fragments P8-F and efALS -F were inserted the vector pLC0 -L simultaneously by using the In-Fusion® HD Cloning Kit ( https://www.takarabio.com ) to generate the plasmid pLC01.

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan). .. The individual luciferase gene was PCR amplified and cloned into pCAMBIA-N-GUS at NcoI/SpeI and PmlI sites by In-Fusion cloning.

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. 3′ End of RLD1 CDS (258 bp), corresponding to the BRX domain, was fused with DNA fragments containing mCherry gene and NOS terminator (mCherry-NOSt ) on the pUC19 vector under the cauliflower mosaic virus 35S promoter (35S:BRXd-mCherry ). mCherry gene without stop codon and LTI6b CDS were amplified by PCR and inserted into the multi-cloning site on the pUC19 vector using In-Fusion HD Cloning Kit (35S:mCherry-LTI6b ).

Purification:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: .. The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. The recombinant plasmids, pET30a-CHH1 and pET30a-CHH2, were transformed into E. coli BL21 (DE3) competent cell for sequencing.

Protein Purification:

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: PAC genes with a histidine-tag at the N-terminal were cloned into pQE-30 vectors for protein purification from Escherichia coli . .. C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. 3′ End of RLD1 CDS (258 bp), corresponding to the BRX domain, was fused with DNA fragments containing mCherry gene and NOS terminator (mCherry-NOSt ) on the pUC19 vector under the cauliflower mosaic virus 35S promoter (35S:BRXd-mCherry ). mCherry gene without stop codon and LTI6b CDS were amplified by PCR and inserted into the multi-cloning site on the pUC19 vector using In-Fusion HD Cloning Kit (35S:mCherry-LTI6b ).

Polyacrylamide Gel Electrophoresis:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. These recombinant proteins were examined by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and quantified to concentration of 500 ng μL–1 by BCA Protein Assay Kit (Tiangen, Beijing) for use.

CRISPR:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: One set was for the expression of CRISPR-Cas13a and the other for the generation of their target RNA. .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a.

Chloramphenicol Acetyltransferase Assay:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. To generate arabinose-inducible target RNA expression vector, firstly we constructed arabinose-inducible RFP expression vector (pKLC-RFP) from pC008-pBR322 by replacing tetracycline-inducible element (pBAD33) and ampicillin resistant gene (bla ) to arabinose-inducible element and chloramphenicol acetyltransferase (CAT).

SDS Page:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. These recombinant proteins were examined by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and quantified to concentration of 500 ng μL–1 by BCA Protein Assay Kit (Tiangen, Beijing) for use.

Plasmid Preparation:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: .. First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. The pC003-LshC2C2 locus included in the plasmid pACYC184 for spacer cloning was a gift from Dr. Feng Zhang (Addgene plasmid #79152 ; RRID:Addgene_79152 ) ( ).

Article Title: APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression
Article Snippet: .. All the amplicons were cloned into the lentiviral plasmid pCSII–CMV–MCS (RIKEN, RDB04377) by using the In-Fusion HD Cloning Kit (TaKaRa), to produce the pCSII–CMV:A3B–3×FLAG–IRES–EGFP donor DNA plasmid ( ). .. Validation of sgRNA targeting efficiency 293T cells were transfected with pSpCas9(BB)–2A–Puro:sgRNA #4 (0.5 μg) using the FuGENE HD Transfection Reagent (Promega).

Article Title: The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata
Article Snippet: The GloSensor-22F cAMP gene (Promega Corporation, Fitchburg, WI, USA), a luciferase-based cAMP reporter, was cloned into a pEBMulti-Neo vector (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for monitoring the cAMP level in HEK293 cells. .. C-terminal deletion mutants of OaPAC and mutants with 15 OaPAC C-terminal residues fused to the C-terminus of bPAC were generated using an In-Fusion HD Cloning kit (TaKaRa, Shiga, Japan).

Article Title: Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells
Article Snippet: .. The Tet-On 3G transactivator of the pTetOne vector (Takara) was PCR-amplified and cloned into the multicloning site of the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara). .. The resulting vector was designated as pCS-CA-Tet3G.

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The insertion sequence was prepared by PCR using the amplified LC sequence as a template and the following primer sets: Forward: CTC GAG CTC AAG CTT GCC GCC ACC ATG GTG CTG, Reverse: CTC GCC CTT GCT CAC TGA TCC TCC TCC TCC TGA.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: .. In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product. .. The plasmid pEGFP-N1 was used as control plasmid.

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: Primers LvCHH1-rpF/rpR, LvCHH2-rpF/rpR were designed with 15 bp extension homologous of linearized pET30a vector (Novogen, France) ends at both ends to amply the ORF of LvCHH1 and LvCHH2 excluding sequence encoding signal peptide, respectively. .. The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States).

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: To generate the enCRISPRa sgRNA vector, the MCP-VP64-IRES-mCherry cassette was PCR amplified from the pJZC34 vector (Addgene, plasmid #62331) and cloned into Bsr GI/Eco RI digested lenti-sgRNA (MS2)-zeo backbone (Addgene, plasmid # 61427). .. Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech).

Article Title: Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate
Article Snippet: .. The fragments P8-F and efALS -F were inserted the vector pLC0 -L simultaneously by using the In-Fusion® HD Cloning Kit ( https://www.takarabio.com ) to generate the plasmid pLC01. .. The plasmids pLC02 and pLC17 were constructed in the same way but with the fragments wxALS -F and llALS -F , respectively.

Article Title: Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system
Article Snippet: .. To construct pCAMBIA-N-GUS, the NOS promoter was amplified from pRI201 and cloned into the pCAMBIA 1305.2 vector (Marker Gene Technologies Inc., Eugene, OR, USA) partially digested with HindIII and XhoI, using an In-Fusion HD Cloning Kit (TaKaRa Bio., Shiga, Japan). .. The individual luciferase gene was PCR amplified and cloned into pCAMBIA-N-GUS at NcoI/SpeI and PmlI sites by In-Fusion cloning.

Article Title: Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control
Article Snippet: .. To construct 35S:RLD1ΔRCC1-GFP and 35S:RLD1ΔFYVE-GFP , upstream region of respective domains in RLD1 CDS and downstream region of the domain were amplified by RT-PCR from the Col wild type, and amplified fragments were inserted between 35S promoter and GFP-NOSt fusion gene on the pUC19 vector using In-Fusion HD Cloning Kit (Clontech). .. 3′ End of RLD1 CDS (258 bp), corresponding to the BRX domain, was fused with DNA fragments containing mCherry gene and NOS terminator (mCherry-NOSt ) on the pUC19 vector under the cauliflower mosaic virus 35S promoter (35S:BRXd-mCherry ). mCherry gene without stop codon and LTI6b CDS were amplified by PCR and inserted into the multi-cloning site on the pUC19 vector using In-Fusion HD Cloning Kit (35S:mCherry-LTI6b ).

Article Title: Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains
Article Snippet: .. The CoreTetCT gene was cloned in plasmid pGEX-6P-1 using an In-Fusion HD Cloning Kit (Clontech, Mountain View, CA). ..

RNA Expression:

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia
Article Snippet: First, the native Cas13a loci including native direct repeats and spacers were isolated from the genome of each Leptotrichia strain by polymerase chain reaction (PCR) and then cloned into the pC003 vector by replacing the shahii Cas13a locus using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan), and then kanamycin resistant gene of the vector was replaced by kanamycin-resistant gene (aphA-3 ) to generate pKLC-Cas13a. .. To generate arabinose-inducible target RNA expression vector, firstly we constructed arabinose-inducible RFP expression vector (pKLC-RFP) from pC008-pBR322 by replacing tetracycline-inducible element (pBAD33) and ampicillin resistant gene (bla ) to arabinose-inducible element and chloramphenicol acetyltransferase (CAT).

Concentration Assay:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: The PCR products were purified and inserted into the linearized pET30a using in-fusion cloning reaction with In-fusion HD Cloning Kit (Clontech, United States). .. These recombinant proteins were examined by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and quantified to concentration of 500 ng μL–1 by BCA Protein Assay Kit (Tiangen, Beijing) for use.

HAT Assay:

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
Article Snippet: Plasmids To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert . .. Then the mCherry cassette was replaced with zsGreen1 by In-Fusion® HD Cloning Kit (Clontech).

CTG Assay:

Article Title: Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG
Article Snippet: .. The sequence of IgG1 LC in the pUC57 vector, a kind gift from Dr. Onitsuka in Tokushima University, was cut out by polymerase chain reaction (PCR) using KOD Plus Neo (Toyobo, Osaka, Japan) and the following primer sets to insert the (G4 S)2 linker: Forward: GCC GCC ACC ATG GTG CTG CAG, Reverse: TGA TCC TCC TCC TCC TGA TCC TCC TCC TCC GCA CTC GCC CCG GTT GAA G. The amplified LC sequence was inserted into the Citrine-N1 vector (Plasmid #54593, Addgene, Cambridge, MA, USA) using an In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). .. The insertion sequence was prepared by PCR using the amplified LC sequence as a template and the following primer sets: Forward: CTC GAG CTC AAG CTT GCC GCC ACC ATG GTG CTG, Reverse: CTC GCC CTT GCT CAC TGA TCC TCC TCC TCC TGA.

Staining:

Article Title: Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei
Article Snippet: In-fusion HD Cloning Kit (Clontech, United States) was used to connect the linearized vector and the PCR product. .. After 48 h culturing, the transfected cells were fixed with 4% paraformaldehyde, stained with 100 ng/ml DAPI (4′, 6-diamidino-2-phenylindole) solution and washed with PBS.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    TaKaRa in fusion pcr cloning kit
    Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two <t>BioBricks</t> (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves <t>PCR,</t> purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.
    In Fusion Pcr Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in fusion pcr cloning kit/product/TaKaRa
    Average 99 stars, based on 1723 article reviews
    Price from $9.99 to $1999.99
    in fusion pcr cloning kit - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.

    Journal: Nucleic Acids Research

    Article Title: In-Fusion BioBrick assembly and re-engineering

    doi: 10.1093/nar/gkq179

    Figure Lengend Snippet: Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.

    Article Snippet: The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit.

    Techniques: Ligation, Plasmid Preparation, Purification, Sequencing, Polymerase Chain Reaction, Amplification, Gel Extraction

    MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the pGL3 -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to PCR. E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: Revelation of p53-independent Function of MTA1 in DNA Damage Response via Modulation of the p21WAF1-Proliferating Cell Nuclear Antigen Pathway *

    doi: 10.1074/jbc.M109.079095

    Figure Lengend Snippet: MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the pGL3 -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to PCR. E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.

    Article Snippet: Mutant mouse p21WAF1 promoter lacking the MTA1 binding region was constructed by cloning the PCR product amplified from the mouse p21WAF1 promoter (region from −1860 to −632) using the forward primer (5′-TAGCCCGGGCTCGAGAGATATCCGTTCGTTCAAACTAAGACTCC-3′) and reverse primer (5′-CCGGAATGCCAAGCTTGAGGCACGAGGGGCGTTACAGGTTCAA-3′), and then cloned into a pGL3 basic vector after digesting with XhoI and HindIII using the Clontech Infusion PCR cloning kit (Clontech).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Purification, Polymerase Chain Reaction

    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Clone Assay, CRISPR, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Expressing

    Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing, Construct

    Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi ( d ) and the expression levels of GFP and RFP were evaluated by western blot analysis ( e ). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading

    Journal: Virology Journal

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

    doi: 10.1186/s12985-017-0776-7

    Figure Lengend Snippet: Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi ( d ) and the expression levels of GFP and RFP were evaluated by western blot analysis ( e ). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading

    Article Snippet: The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan).

    Techniques: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, Quantitative RT-PCR, In Vivo, Produced, Reverse Transcription Polymerase Chain Reaction, Amplification, Activity Assay, Construct, Fluorescence, Microscopy, Western Blot

    Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR ( d ), or 35S-HH3-SYNV or 35St-SYNV ( e ), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA

    Journal: Virology Journal

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

    doi: 10.1186/s12985-017-0776-7

    Figure Lengend Snippet: Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR ( d ), or 35S-HH3-SYNV or 35St-SYNV ( e ), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA

    Article Snippet: The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan).

    Techniques: Expressing, Microscopy, Western Blot, Staining, Northern Blot, Quantitative RT-PCR, Isolation