Human LAG3 and mouse Lag3 cDNA clones were kindly obtained from Dr. Charles Drake at the Johns Hopkins University School of Medicine. Lag3 deletion mutants (D1, D2, D3, D4, TM, and ICD) with a HA tag and deletion mutants of Lag3 D1 domain with a myc tag were constructed by PCR with In‐Fusion HD Cloning Plus systems (Takara; Bio Inc., Otsu, Japan) using the CloneAmp HiFi PCR Premix (a high‐fidelity PCR polymerase included with all In‐Fusion HD Cloning Plus systems), and primers with a 15 bp overlap at their 5′ ends to incorporate the mutation of interest. The DNA was separated on a 1% agarose gel, and the appropriate band was excised and isolated using a gel extraction kit (Qiagen, Valencia, CA, USA). In‐Fusion HD enzyme premix was added to the linearized PCR product and transformed into the Stellar Competent Cells (Takara; Bio Inc., Otsu, Japan). The integrity of plasmid sequences was verified through sequencing.
In fusion hd cloning plus system
Manufactured by Takara Bio
Sourced in Japan
The In-Fusion HD Cloning Plus System is a cloning kit designed for rapid and efficient DNA fragment assembly. It enables the seamless joining of multiple DNA fragments, regardless of their source or size, in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Stellar competent cells, by Takara Bio (3 mentions)
Gel extraction kit, by Qiagen (2 mentions)
Cloneamp hifi pcr premix, by Takara Bio (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
Qiaprep spin miniprep, by Qiagen (1 mentions)
Spectinomycin, by Merck Group (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Glomax microplate luminometer, by Promega (1 mentions)
11 protocols using in fusion hd cloning plus system
1
Generating LAG3 Deletion Mutants
2
Generating an In-Frame Deletion Plasmid
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To construct the in-frame deletion of pp2 (OG1RF 11 046–11063), regions approximately 1Kb upstream and downstream of the genes were amplified from OG1RF using primer pairs pp2.infu.1F/R and pp2.infu.2F/R for upstream and downstream regions, respectively (Table S1, Supporting Information). These products were sewn together and amplified using pp2.infu.1F/pp2.infu.2R. The temperature sensitive plasmid pGCP213 was amplified using the pGCPinphuF/R primer pair. The pp2.infuF/R PCR product of approximately 2Kb was then cloned into the pGCP213 fragment using the In-Fusion HD Cloning Plus System (Takara Bio Inc, Shimogyō-ku, Kyoto) to generate the temperature sensitive deletion plasmid pGCPpp2.
Deletion constructs were then transformed into OG1RF by electroporation and the transformants were selected at 30°C on agar plates with kan. Chromosomal integrants were selected by growth at 42°C on the agar plate in the presence of kan. Selection for excision of the integrated plasmid by homologous recombination was accomplished by growing the bacteria at 30°C in the absence of kan in the broth. Loss of the pp2 locus in kanamycin-sensitive bacteria was demonstrated by PCR using primer pair infu_check_pp2F/R.
Deletion constructs were then transformed into OG1RF by electroporation and the transformants were selected at 30°C on agar plates with kan. Chromosomal integrants were selected by growth at 42°C on the agar plate in the presence of kan. Selection for excision of the integrated plasmid by homologous recombination was accomplished by growing the bacteria at 30°C in the absence of kan in the broth. Loss of the pp2 locus in kanamycin-sensitive bacteria was demonstrated by PCR using primer pair infu_check_pp2F/R.
3
Cloning Gene Construct into pTT3 Vector
4
Cloning and Expression of Recombinant hmAbs
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Cloning of the variable regions of IgG heavy and light chains (VH; and VL = κ/λ) into relevant AbVec IgG expression vectors and subsequent expression of hmAbs was performed as previously described (22 (link)). A slight modification to the protocol involved substituting restriction endonuclease cloning with a ligation-independent method using the InFusion HD Cloning Plus system (Takara Bio) for enhanced cloning efficiency. The sequences of all V gene fragments were obtained by Sanger sequencing and analysed using the IGBLAST tool hosted on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/igblast/ ). Calculations of the replacement-to-silent mutation ratios (dN/dS) were performed using approximate methods (23 (link)). Thresholds of >2.9 and >1.5 are indicative of positive antigen-driven selection for replacement mutations for CDR and FR regions, respectively (24 (link)).
5
UHRF Protein Modification Protocol
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Amino acid UHRF point mutants were created using the In-Fusion HD Cloning Plus System (Takara) or ordered from Genscript. The In-Fusion HD Cloning Plus System was also used to generate UHRF2 TTD Δ stretch and UHRF2 TTD-PHD Δ stretch constructs, which have amino acids 141-193 removed from the coding sequence. UHRF1 TTD + stretch was created by adding amino acids 141-193 from UHRF2 to UHRF1 TTD between amino acids 167 and 168.
6
Construction of pp2 Gene Deletion Mutant
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To construct the in-frame deletion of pp2 (OG1RF 11046-11063), regions approximately 1Kb upstream and downstream of the genes were amplified from OG1RF using primer pairs pp2.infu.1F/R and pp2.infu.2F/R for upstream and downstream regions, respectively (Table S1). These products were sewn together and amplified using pp2.infu.1F / pp2.infu.2R. The temperature sensitive plasmid pGCP213 was amplified using the pGCPinphuF/R primer pair. The pp2.infuF/R PCR product of approximately 2Kb was then cloned into the pGCP213 fragment using the In-Fusion HD Cloning Plus System (Takara Bio Inc, Shimogyō-ku, Kyoto) to generate the temperature sensitive deletion plasmid pGCPpp2. Deletion constructs were then transformed into OG1RF by electroporation and the transformants were selected at 30°C on agar plates with kan. Chromosomal integrants were selected by growth at 42°C on the agar plate in the presence of kan.
Selection for excision of the integrated plasmid by homologous recombination was accomplished by growing the bacteria at 30°C in the absence of kan in the broth.
Loss of the pp2 locus in kanamycin-sensitive bacteria was demonstrated by PCR using primer pair infu_check_pp2F/R
Selection for excision of the integrated plasmid by homologous recombination was accomplished by growing the bacteria at 30°C in the absence of kan in the broth.
Loss of the pp2 locus in kanamycin-sensitive bacteria was demonstrated by PCR using primer pair infu_check_pp2F/R
7
Amplification and Cloning of Membrane Protein Genes
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Genes encoding the membrane proteins were amplified from genomic DNA using iProof High-Fidelity DNA Polymerase (Bio-Rad #172-5302). Francisella tularensis subsp. tularensis SCHU S421 (link) genomic DNA was provided by Drs. C. Rick Lyons, Terry H. Wu, and Jason Zsemlye (University of New Mexico). ASFV genomic DNA from the highly virulent isolates Georgia 2007/152 (link) and Malawi Lil 20/153 (link) was provided by Dr. Linda K. Dixon (The Pirbright Institute, United Kingdom). Unless noted, cloning was achieved using the In-Fusion HD Cloning Plus system (Clontech #638910). This ligation-independent system allows simple matching between 15 base pairs of each end of a PCR-derived insert with the ends of the linearized parent vector. Plasmid DNA was prepared with the QIAprep Spin Miniprep or Plasmid Maxi system (QIAGEN #27106 and #12163, respectively). DNA elutions were done using molecular grade water. Sequence confirmation of the complete insert in each plasmid clone was performed at the School of Life Sciences DNA Laboratory at Arizona State University. Vectors used in this study and representative primers are in Supplementary Tables 2 and 3, respectively.
8
Cloning pPIGS:GFP-PIGS Construct
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The pPIGS:GFP-PIGS construct was created by overlap PCR with PrimeSTAR® GXL DNA Polymerase (TaKaRa Bio Inc.; Catalog # R050A) and DNA templates and primers listed in Additional Table 1 and cloned into pH7WG plasmid linearized with SalI-HF (NEB, Cataog # R3138S) and AscI (NEB, Catalog # R0558S) by using the In-Fusion HD Cloning Plus system (Clontech, Catalog # 639645). The recombinant plasmids were transformed into Stellar™ Competent Cells (Clontech, Catalog # 636763), and positive colonies were selected on LB plates containing spectinomycin (100μg/mL, Sigma-Aldrich, Catalog # 85555). The construct was sequence verified (Eton Bioscience, Inc.) before transforming into Agrobacterium tumefaciens (GV3101 pMP90 strain). The positive colony selected for transforming into Arabidopsis was also verified by colony PCR for the presence of the transgene.
9
Assessing miR-26a binding to MAPK6 3'UTR
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MAPK6 3′-UTR fragments with wild type (WT) or mutant miR-26a binding sites were cloned by PCR. The primers are as follows:
WT MAPK6 3′-UTR
Forward primer: 5′-AACGAGCTCGCTAGCCTCGAGTCATGAAATGTGTTGTGTCTT-3′
Reverse primer: 5′-CCTGCAGGTCGA CTCTA ACACCATTAAGACTTCTAGGAGC-3′
mutant MAPK6 3′-UTR
Forward primer: 5′-AACGAGCTCGCTAGCCTCGAGTTTCCGTCCAAATCAGAAGGTGT-3′
Reverse primer: 5′-CCTGCAGGTCGACTCTAGACACCATTAAGACTTCTTCCTCG-3′
PCR products were inserted into the pmirGLO vector (Promega) with an In-Fusion HD Cloning Plus system (Clontech). MiRNA target reporter assays were performed in quadruplicate in 96-well plates. Then, 200 ng of each construct or vehicle control was co-transfected with miR-26a agomir or negative control (50 nM) into HEK293 cells using Lipofectamine 2000 according to the recommended protocol. Firefly and Renilla luciferase activities were sequentially measured using the Dual-Glo™ Luciferase Assay system (Promega) with a GloMax microplate luminometer (Promega) 48 h after transfection. Relative luciferase activity was normalized with Renilla luciferase activity.
WT MAPK6 3′-UTR
Forward primer: 5′-AACGAGCTCGCTAGCCTCGAGTCATGAAATGTGTTGTGTCTT-3′
Reverse primer: 5′-CCTGCAGGTCGA CTCTA ACACCATTAAGACTTCTAGGAGC-3′
mutant MAPK6 3′-UTR
Forward primer: 5′-AACGAGCTCGCTAGCCTCGAGTTTCCGTCCAAATCAGAAGGTGT-3′
Reverse primer: 5′-CCTGCAGGTCGACTCTAGACACCATTAAGACTTCTTCCTCG-3′
PCR products were inserted into the pmirGLO vector (Promega) with an In-Fusion HD Cloning Plus system (Clontech). MiRNA target reporter assays were performed in quadruplicate in 96-well plates. Then, 200 ng of each construct or vehicle control was co-transfected with miR-26a agomir or negative control (50 nM) into HEK293 cells using Lipofectamine 2000 according to the recommended protocol. Firefly and Renilla luciferase activities were sequentially measured using the Dual-Glo™ Luciferase Assay system (Promega) with a GloMax microplate luminometer (Promega) 48 h after transfection. Relative luciferase activity was normalized with Renilla luciferase activity.
10
Endogenous Tagging of TGGT1_228170 via LIC
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For C-terminal endogenous tagging of TGGT1_228170, the 3′ region directly upstream of the stop codon was amplified from RHΔku80 parasite genomic DNA by PCR and inserted into the pLIC-3×HA-DHFR (45 (link)) vector at the PacI restriction site by ligation-independent cloning (LIC), facilitated by use of an InFusion HD Cloning Plus system (Clontech). The sequences of the primers for this and all reactions used in this work are presented in Table S3 in the supplemental material. Fifty micrograms of XcmI-linearized vector was transfected into RHΔku80 parasites, and the resultant population was selected for the presence of the pyrimethamine-resistant dihydrofolate reductase (DHFR) allele, which is included in the vector (46 (link)). Independent clones were established by limiting dilution of the transfected population and confirmed by immunofluorescence assay and Western blotting.
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