Full‐length OsTF1L and 3‐kb upstream sequences of target genes were amplified by PCR using high‐fidelity DNA polymerase PrimeStar (TaKaRa, Tokyo, Japan). OsTF1L was cloned into linearized pHBT vector containing a 35S promoter by BamHI and PstI restriction enzyme using In‐Fusion HD cloning system (TaKaRa). Target genes promoter region were cloned into linearized pGST6‐LUC‐NOS vector harbouring a GST6 enhancer/promoter‐FIREFLY LUCIFERASE by BamHI and NcoI restriction enzyme using In‐Fusion HD cloning system (TaKaRa). 15 μL of vector solution including 3 μg of effector, 1 μg of reporter and 0.5 μg of internal control was transformed into isolated rice protoplasts using PEG (polyethylene glycol)‐mediated transformation (Jung et al., 2015 ). The Dual‐Luciferase Reporter Assay System (Promega, Fitchburg, WI) was used to measure the luciferase activity according to manufacturer's manual, and the fluorescent values of luciferase were detected with an Infinite M200 System (Tecan, Seestrasse, Männedorf, Switzerland). Three independent transfections for each sample were performed, and the value of firefly luciferase was normalized to that of renilla luciferase. The 35S::rLUC was used as an internal control.
In fusion hd cloning system
Manufactured by Takara Bio
Sourced in United States, Japan
The In-Fusion HD cloning system is a DNA assembly method that enables the seamless joining of multiple DNA fragments in a single, isothermal reaction. The system utilizes proprietary enzyme mixes to efficiently ligate DNA fragments with overlapping ends, facilitating the construction of recombinant plasmids.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (4 mentions)
Pmirglo, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Psicheck 2 plasmid, by Promega (2 mentions)
Dual luciferase assay, by Promega (2 mentions)
Gel extraction kit, by Qiagen (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Cloneamp hifi pcr premix, by Takara Bio (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Bglii, by New England Biolabs (2 mentions)
Dna ligation kit, by Takara Bio (2 mentions)
Pir2 cells, by Thermo Fisher Scientific (2 mentions)
E coli stellar cells, by Takara Bio (2 mentions)
Plenti cmv to hygro, by Addgene (2 mentions)
Tet on 3g inducible expression system, by Takara Bio (2 mentions)
Mln4924, by Cayman Chemical (2 mentions)
Accuprime pfx, by Thermo Fisher Scientific (2 mentions)
Pureyield plasmid midiprep system, by Promega (2 mentions)
Tetone inducible expression system, by Takara Bio (1 mentions)
Xfect transfection reagent, by Takara Bio (1 mentions)
79 protocols using in fusion hd cloning system
1
Transcription Factor Regulation Assay
2
Cloning and Mutagenesis of Promoter Regions
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DNA fragments of the promoter region of HEMA1(1.7 kb), GUN4(1.5 kb), CAO (1.2 kb) and BPG4 (2.5 kb) were amplified and cloned into a pGLHNew_RLH vector obtained from Dr. Nobutaka Mitsuda by an In-Fusion HD cloning system (Takara). The G-box (CACGTG) sequences in the BPG4 promoter region were mutated to AAAAAA by inverse PCR using PrimeSTAR Max (Takara). The full-length BES1, VP64, BPG4, and GLK1 CDSs were cloned into a pENTR/D-TOPO vector (Invitrogen) and subsequently cloned via LR recombination into a binary Gateway expression vector pDEST-35SHSP obtained from Dr. Nobutaka Mitsuda. To generate the 35S:BES1-VP64 effector, DNA fragments of VP64 were amplified and cloned into the 35S:BES1 effector by an In-Fusion HD cloning system (Takara). The 35S:GFP effector was obtained from Dr. Nobutaka Mitsuda. The reporter construct was cotransformed together with the effectors into Col-0 protoplasts for the transcriptional activity assay described previously94 ,95 (link). The ratios of LUC to REN activity were calculated to define the relative promoter activity. To detect the LUC and REN activity, a Pickagene® Dual Sea Pansy Luminescence Kit (Toyo Bnet, Tokyo, Japan) was used. Data were obtained from four replicates, and the primers used are listed in Supplementary Table S8 .
3
Recombinant Protein Production of BPG4 and GLK1
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Production of MBP-BES1 DBD was performed as described previously33 (link). The BPG4 full-length CDS was cloned into a pMAL-c4x vector (NEB, Ipswich, MA, USA) by an In-Fusion HD cloning system (Takara). For recombinant protein production, pMAL-c4x-BPG4 was transformed into Escherichia coli BL21 (DE3) pLysS. Expression was induced with 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) at 37 °C for 4 h. MBP-BPG4 was subsequently purified using amylose resin (NEB). The production of GST-GLK1 was performed as described previously, with minor modifications91 (link). The GLK1 full-length CDS was cloned into a pGEX-6P-3 vector by an In-Fusion HD cloning system (Takara). For recombinant protein production, the pGEX-6P-3-GLK1 vector was transformed into Escherichia coli Rosetta cells (DE3). Expression was induced with 0.5 mM IPTG at 16 °C overnight. GST-GLK1 was purified using Glutathione SepharoseTM 4B (GE Healthcare, Chicago, IL, USA). GST-GLK1 and MBP-BPG4 were concentrated and desalinated using a Vivaspin 20 Centrifugal Concentrator (Sartorius, Göttingen, Germany). The primers used are listed in Supplementary Table S6 .
4
Generation of Inducible APOL1 Cell Line
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The WT APOL1 addback cell line was generated using TetOne Inducible Expression System (Clontech, Mountain View, USA) following manufacturer’s instructions. Briefly, APOL1 was PCR-amplified using primers 5′-CCCTCGTAAAGAATTCATGTCAGAGGAAGCTGGAGCGAGG-3′ and 5′-GCAGAGATCTGGATCCTCACAGTTCTTGGTCCGCCTGCAG-3′ and cloned into pTetOne vector using In-Fusion® HD Cloning System (Clontech). The pTetOne vector was transfected into podocytes using Xfect transfection reagent (Clontech). Hygromycin resistant cells were screened for APOL1 induction following doxycycline induction.
5
Cloning CLEC18A with 2xHA and eGFP
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Cloning steps described in this section followed procedures outlined for the In-Fusion® HD Cloning system (Clontech, Mountain View, CA). Full primer sequences are listed in Table 1 .
To generate the pMOS1_AePUb-CLEC18A-2xHA_3xp3-eGFP plasmids, the coding sequence of CLEC18A was amplified from the pMACS Kk.HA(C)-CLEC18A plasmid (49 (link)) by PCR using the primer pair pAc5.1_3xHA_fusion_CLEC18A-F and pAc5.1_3xHA_fusion_CLEC18A-R. PCR-amplified DNA fragments were inserted into the SphI/XhoI site of the pAePUb_3xHA vector to create a pAePUb_CLEC18A_3xHA transition plasmid. The transition plasmid pAePUb_CLEC18A_3xHA was then used as a template to amplify the CLEC18A-2xHA DNA fragment via PCR using the primer pair pMOS1_AePUb_fusion_CLEC18A-2xHA-F and pMOS1_AePUb_fusion_CLEC18A-2xHA-R. The CLEC18A-2xHA PCR product was subcloned into the BglII/XhoI sites of the pMOS1-AePUb_Den3-4miR_3xp3-eGFP vector to create pMOS1_AePUb-CLEC18A-2xHA_3xp3-eGFP constructs, which were then used as donor plasmids for embryo microinjection.
To generate the pMOS1_AePUb-CLEC18A-2xHA_3xp3-eGFP plasmids, the coding sequence of CLEC18A was amplified from the pMACS Kk.HA(C)-CLEC18A plasmid (49 (link)) by PCR using the primer pair pAc5.1_3xHA_fusion_CLEC18A-F and pAc5.1_3xHA_fusion_CLEC18A-R. PCR-amplified DNA fragments were inserted into the SphI/XhoI site of the pAePUb_3xHA vector to create a pAePUb_CLEC18A_3xHA transition plasmid. The transition plasmid pAePUb_CLEC18A_3xHA was then used as a template to amplify the CLEC18A-2xHA DNA fragment via PCR using the primer pair pMOS1_AePUb_fusion_CLEC18A-2xHA-F and pMOS1_AePUb_fusion_CLEC18A-2xHA-R. The CLEC18A-2xHA PCR product was subcloned into the BglII/XhoI sites of the pMOS1-AePUb_Den3-4miR_3xp3-eGFP vector to create pMOS1_AePUb-CLEC18A-2xHA_3xp3-eGFP constructs, which were then used as donor plasmids for embryo microinjection.
6
Recombinant Human Cytochrome P450 Production
7
Insect Odorant Receptor Plasmid Cloning
8
Recombinant Protein Purification of Os1348
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The fragment carrying 1348 was amplified from the genomic DNA of Pseudomonas sp. Os17 by PCR with the primers 25F1348 and 25R1348 (Supplementary Table S2 ). The PCR product was inserted into the NdeI/BamHI sites of the expression vector pET25b (+) (Novagen) with the In-Fusion HD Cloning system (Clontech). The resultant plasmid, pET25-1348, was introduced into E. coli BL21 (DE3) (Merck) and the strain was grown at 37°C. The expression of the recombinant Os1348 protein was induced by the addition of isopropyl-β-D -thiogalactopyranoside at a final concentration of 0.4 mM and growth continued at 16°C for 16 h. Harvested cells were resuspended in PBS (pH 7.4) and lysed by sonication. Debris was removed by centrifugation at 40,000 × g for 30 min. The lysate was applied to a 5-ml HiTrap SP HP column (GE Healthcare). The column was washed with 20 mM sodium acetate (pH 5.2) and eluted with a linear gradient of 0–500 mM NaCl. Os1348 fractions were purified further on HiLoad 26/60 Superdex75 prep grade (GE Healthcare) pre-equilibrated with buffer consisting of 10 mM Tris–HCl (pH 7.5) and 150 mM NaCl. After dialysis at 4°C for 16 h against 20 mM sodium acetate (pH 5.2), the supernatant was purified by a Mono S 10/100 GL column (GE Healthcare) and a 0–300 mM NaCl elution gradient.
9
Mutagenesis of GFAP Protein Isoforms
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GFAP mutations were introduced by site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) using the human wild-type GFAP in the pcDNA 3.1(−) vector (Thermo Fisher Scientific, Waltham, MA) as a template (Perng et al., 2006 (link)). The mutagenic primers used for the construction of GFAP mutants were as follows:
R88C GFAP forward, 5′-ATCGAGAAGGTT
R88C GFAP reverse, 5′-GTTCCAGGAAGC
∆4GFAP (a splice site mutation leading to skipping of exon 4) forward, 5′-AGGAAGATCCACGAGGAGTTTGCAGACCTGACAGACGCTGCT-3′
∆4GFAP reverse, 5′-AGCAGCGTCTGTCAGGTCTGCAAACTCCTCGTGGATCTTCCT-3′
IDF GFAP (deletion and insertion mutations leading to a frameshift of GFAP) forward, 5′-AGCAGGAGCACAAGGATGATCGGCAGGACCCACCTG-3′
IDF GFAP reverse, 5′-GATTTGGGTCCTGCCTCATGAGACGGGGCAGAGGCC-3′
10
Molecular Biology Techniques for Fungal DNA
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DNA isolation and manipulation were performed with a standard method59 . Genomic DNA was isolated from M. oryzae with a DNeasy plant total DNA isolation kit (Qiagen, Valencia, CA, USA). DNA fragments were isolated from agarose with a gel extraction kit (Qiagen). Plasmids were purified with a QIAprep Spin Miniprep kit (Qiagen). PCR was carried out with a DNAEngine Peltier thermal cycler (Bio-Rad, Hercules, CA, USA). DNA amplification via PCR was conducted with KOD-Plus-neo DNA polymerase (Toyobo, Osaka, Japan). DNA fragment ligation and circularization for vector construction was conducted via infusion reaction using an In-Fusion HD cloning system (Clontech, Palo Alto, CA, USA). DNA sequencing was carried out with a BigDye terminator ver3.1 kit (Applied Biosystems, Foster City, CA, USA). Sequencing products were run on an automated 3730 × l capillary DNA analyzer (Applied Biosystems).
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