Vector nti advance

Manufactured by Thermo Fisher Scientific

Sourced in United States

Vector NTI Advance is a comprehensive software suite for molecular biology and bioinformatics applications. It provides tools for DNA and protein sequence analysis, primer design, virtual cloning, and bioinformatics data management.

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23 protocols using vector nti advance

Cloning and Sequencing of Date Palm CLO Genes

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Full-length ORFs of PdCLO2 and PdCLO4 genes (720 and 711 bp, respectively) were amplified by PCR using date palm cDNA. For further purification of recombinant proteins, primers were designed to add a His tag at the N-terminal ends of the PdCLO2 and PdCLO4 genes (Table S1). Purified PdCLO2 and PdCLO4-PCR products were ligated to pGEM-T Easy vector (Promega, USA) following the manufacturer's manual. The recombinant plasmids pGEM-T/PdCLO2 and pGEM-T/PdCLO4 were transferred into E. coli Top10 competent cells. Recombinant plasmids were extracted from E. coli Top10 using a plasmid purification mini kit (Qiagen, Germany). The presence of PdCLO2 and PdCLO4 genes in the isolated plasmid was confirmed by PCR and SacI-EcoRI digestion. At least three clones of both genes were sequenced on both strands using T7 and SP6 primers on an ABI 310 Genetic Analyser (Applied Biosystems) using Big Dye Terminator kit (Applied Biosystems). Nucleotide sequence analysis and multiple alignments of the nucleotide and deduced amino acid sequences were performed with Vector NTI advance (Invitrogen).

Hanano A., Almousally I., Shaban M., Rahman F., Hassan M, & Murphy D.J. (2017). Specific Caleosin/Peroxygenase and Lipoxygenase Activities Are Tissue-Differentially Expressed in Date Palm (Phoenix dactylifera L.) Seedlings and Are Further Induced Following Exposure to the Toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin. Frontiers in Plant Science, 7, 2025.

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Protein Sequence Alignment of MARCKS

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The amino acid sequence alignment of members of the MARCKS protein family was generated by AlignX, a multiple alignment algorithm embedded in the Vector NTI Advance software suite (Version 11.0; Invitrogen). The alignment was based on the blosum62mt2 score matrix and was computed with gap opening and extension penalties set to 10 and 0.05, respectively.

Mehrabian M., Brethour D., Williams D., Wang H., Arnould H., Schneider B, & Schmitt-Ulms G. (2016). Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue. PLoS ONE, 11(6), e0156779.

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Identification of DNA Polymorphisms in Brassica Genes

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To assess for DNA polymorphisms, sequences for BnaFLC.A02 and BnaFT.A02 were extracted from the Cabriolet and Darmor sequence PileUp and aligned against the Darmor‐bzh BnaFLC.A02 and BnaFT.A02 sequences (Chalhoub et al., 2014), downloaded from plants.ensembl.org. Sequence polymorphisms were identified, and amino acid sequence changes were predicted using AlignX (Vector NTI Advance®, Invitrogen^TM, now Thermo Fisher Scientific, https://www.thermofisher.com). To confirm the presence of the polymorphisms in Cabriolet and Darmor, primers were designed to amplify and sequence regions of the BnaFLC.A02 and BnaFT.A02 genes from both varieties (Table S3). DNA was isolated from both varieties using the Edwards DNA extraction method (Edwards et al., 1991), and PCR was performed using AmpliTaq Gold^TM DNA polymerase (Applied Biosystems^TM, now Thermo Fisher Scientific) according to the manufacturer's instructions with an annealing temperature of 58 °C. PCR products were prepared for sequencing using the Big Dye V3.1^TM terminator protocol (Applied Biosystems^TM, now Thermo Fisher Scientific), and capillary sequencing was performed by Eurofins Genomics, EU. Sequences were aligned and analysed using AlignX.

Tudor E.H., Jones D.M., He Z., Bancroft I., Trick M., Wells R., Irwin J.A, & Dean C. (2020). QTL‐seq identifies BnaFT.A02 and BnaFLC.A02 as candidates for variation in vernalization requirement and response in winter oilseed rape (Brassica napus). Plant Biotechnology Journal, 18(12), 2466-2481.

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Genomic Sequence Analysis of Pseudomonas aeruginosa

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The genomic sequence of P. aeruginosa PAO1 was obtained from the Pseudomonas genome database (www.pseudomonas.com) and Kyoto Encyclopedia of Genes and Genomes (www.genome.jp/kegg/). Nucleotide and protein sequences were compared using BLASTn and BLASTp, respectively; both programs were provided by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Restriction sites and oligonucleotide primer sequences were identified using Vector NTI Advance^® software (Invitrogen). Promoter regions were predicted using BPROM software (Softberry, Inc).

Lo Y.L., Shen L., Chang C.H., Bhuwan M., Chiu C.H, & Chang H.Y. (2016). Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1. PLoS ONE, 11(5), e0155397.

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Protein Quantification and Alignment

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SDS-PAGE was performed using 4-12% polyacrylamide pre-cast gels (Novex NuPAGE; Invitrogen) with MOPS running buffer (Invitrogen). EZ-Run™ Rec Protein Ladder was from Fisher. Gels were stained with SimplyBlue Safe Stain (Invitrogen). Protein concentrations were determined using protocols from Bradford [22 (link)] using bovine serum albumin as the standard. Protein alignments were prepared using Vector NTI Advance ^® (Invitrogen).

Hu Y., Palmer S.O., Munoz H, & Bullard J.M. (2015). High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae. Current drug discovery technologies, 11(4), 279-292.

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Bioinformatic Analysis of EaZIP Homologs

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All nucleotide and protein sequences homologous to EaZIP of ‘Golden Pothos’ were obtained by searching NCBI GenBank databases (http://www.ncbi.nlm.nih.gov/genbank/). Sequence alignment analysis was conducted by using the Vector NTI Advance^® software version 11.5 (InVitrogen, Carlsbad, CA, U.S.A.). A web-based analysis tool ChloroP was used to verify cleavage site position and sequence length of cTP of all EaZIP-related proteins (http://www.cbs.dtu.dk/services/ChloroP/) [36 ].

Guan X., Li Z., Zhang Z., Wei X., Xie J., Chen J, & Chen Q. (2017). Overexpression of an EaZIP gene devoid of transit peptide sequence induced leaf variegation in tobacco. PLoS ONE, 12(4), e0175995.

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Bioinformatic Analysis of DH-PBAN Genes

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The obtained raw sequence data were edited and assembled into contigs with ContigExpress of the Vector NTI Advance® program (Invitrogen, Carlsbad, CA). Nucleic acid sequences were translated into amino acid sequences, and open reading frames (ORFs) were identified using DNAsmac (http://biofreesoftware.com/dnasmac). Each sequence segment was aligned using the ClustalX2 application [34] (link). To determine intron-exon boundaries and intron phases, the Marvi-DH-PBAN cDNA sequence was subjected to ClustalX2 against the corresponding genomic DNA sequence. The last two nucleotides of an exon in a cDNA sequence are usually AG (adenine-guanine), the “shadow sequence” [35] (link), [36] (link). These nucleotides are often mistakenly aligned with the last two bases of the intronic acceptor site, which are also AG in spliceosomal introns as a general rule, but other misalignment problems are not uncommon. Therefore, the intron phases were verified manually by inserting the intron position right before the upstream intronic donor base pair, GT (guanine-thymine), or immediately after AG at the end of the preceding exon. The evolutionary tree for interspecific and intraspecific phylogenetic relationships between DH-PBAN genes on the protein level was produced with MEGA5 [37] (link).

Chang J.C, & Ramasamy S. (2014). Identification and Expression Analysis of Diapause Hormone and Pheromone Biosynthesis Activating Neuropeptide (DH-PBAN) in the Legume Pod Borer, Maruca vitrata Fabricius. PLoS ONE, 9(1), e84916.

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Cx26 Cloning and Mutagenesis Protocol

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Wild type rat Cx26 cDNA (accession: NM_001004099) was cloned in the pcDNA3.1/CT-GFP-TOPO vector (Invitrogen). Site-directed mutagenesis was generated using the Quick-Change II kit (Agilent Technologies). For some experiments Cx26 or its variants contain hemagglutinin epitope tag appended to their C-terminus. Primers sequences are detailed in supplementary Table S2. DNA was sequenced by Macrogen Inc (Seoul, Korea), and analyzed using the “Vector NTI Advance” software (Invitrogen).

García I.E., Maripillán J., Jara O., Ceriani R., Palacios-Muñoz A., Ramachandran J., Olivero P., Pérez-Acle T., González C., Sáez J.C., Contreras J.E, & Martínez A.D. (2015). Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43. The Journal of investigative dermatology, 135(5), 1338-1347.

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Quantitative RT-PCR Analysis of Mouse Cardiomyocytes

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Total RNA was extracted from isolated mouse LV myocytes utilizing TRIZOL reagent (Invitrogen). cDNA was obtained from 1 µg total RNA using MultiScribe reverse transcriptase kit (Applied Biosystems). Real-time RT-PCR was performed with primers designed using the Vector NTI Advance 11 (Invitrogen) software^{12 (link),55 (link),64 (link)}. The sequences of primers are indicated in Supplementary Table 1. The StepOnePlus Real-Time PCR system (Applied Biosystems) was employed for quantitative RT-PCR. In each case, cDNA was combined with Power SYBR Green Master Mix (Applied Biosystems) in a 10 µl reaction. Cycling conditions were as follow: 95°C for 10 min followed by 40 cycles of amplification (95°C denaturation for 15 sec, 60°C annealing and extension for 1 min). The melting curve was then obtained. C_t values were normalized with respect to β-2-microglobulin (β2m). To avoid the influence of genomic contamination, forward and reverse primers for each gene were located in different exons. PCR products were run on 3% agarose/1x TBE gel to confirm the specificity of the reaction. Total RNA extracted from mouse brain and skeletal muscle was employed as controls. For fibrotic markers^{18 (link)}, RNA was extracted from LV tissue. C_t values were normalized with respect to hypoxanthine guanine phosphoribosyl transferase (Hprt)^{18 (link)}.

Signore S., Sorrentino A., Borghetti G., Cannata A., Meo M., Zhou Y., Kannappan R., Pasqualini F., O'Malley H., Sundman M., Tsigkas N., Zhang E., Arranto C., Mangiaracina C., Isobe K., Sena B.F., Kim J., Goichberg P., Nahrendorf M., Isom L.L., Leri A., Anversa P, & Rota M. (2015). Late Na+ current and protracted electrical recovery are critical determinants of the aging myopathy. Nature Communications, 6, 8803.

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Establishing Lentiviral CRISPR Knockout Clones

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The following gRNA sequences were cloned into the pNGx-LV-g003 lentiviral backbone (DeJesus et al., 2016 (link)) and transduced into the HCT116-Cas9 clone described above at an MOI 0.5: ATP2C1 5’- GAACTCTATCCCCAACAGAA-3’, FERMT2 5’-GGTGGGAAAAGAAGAGAACT-3’, DUSP5 5’- GCGCTACGTGCTGCCCGACG-3’. The cells were selected for 4 days using 2 μg/ml puromycin and transduction efficiency (RFP signal) was assessed by FACS analysis. Selected cells were diluted to a density of 100-300cells / 20 ml and seeded in 15 cm dishes in puromycin containing medium. After 2 weeks, single colonies were picked using cloning discs, expanded for another 10-15 days and frozen down as master stocks.
Editing was assessed by genotyping as follows. Genomic DNA was extracted for each clone using Qiagen’s QIAamp DNA mini Kit (51304) and the DNA regions across each sgRNA were amplified by PCR using Promega’s GoTaqGreen master mix (M7122) following the standard protocol. The PCR bands were extracted using Zymo Research’s Zymoclean Gel DNA Recovery Kit (D4001) and submitted to Sanger sequencing (Microsynth standard service). The sequences were analysed using the Vector NTI Advance (Invitrogen, version 11.5.4).

Estoppey D., Schutzius G., Kolter C., Salathe A., Wunderlin T., Meyer A., Nigsch F., Bouwmeester T., Hoepfner D, & Kirkland S. (2021). Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity. iScience, 24(11), 103323.

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