All SNCG gene identification sequence data is based on http://ensemble.org with gene accession number: ENSG00000173267. Unidentified PCR amplicons were verified by DNA sequencing. The specific novel cDNA was cloned into pJET1.2/blunt Cloning Vector using Clone Jet Kit (Thermo) according to manufacturer's protocol. Commercial DNA sequencing was carried out by GATC biotech (European Genome and Diagnostics Centre, Konstanz, Germany). For recombinant protein expression of isoform 2 short specific primers binding at the beginning of exon 1 (forward primer 5′-GCC TGC AGC AGC ACA ACC-3′) or at the end of exon 5 (reverse primer 5′-TTC TCG AGC AGG AGT GGG CTC AAG T-3′), respectively, were used to create full-length cDNA samples of the isoform. CDNA samples were used as templates to clone isoform 2 short sequence into the expression vector pCMV Script Vector (Agilent Technologies, Stratagene®, Santa Clara, USA). EcR V and Xho I were used as cloning sites. Vector inserts were verified using T3 promotor primers Vector (Agilent Technologies, Stratagene®) and insert specific primers (Apara).
Clonejet kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CloneJET kit is a molecular biology tool used for DNA cloning. It provides all the necessary reagents and protocols to perform efficient and reliable DNA fragment insertion into bacterial plasmid vectors.
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Lab products found in correlation
Stbl3 bacteria, by Thermo Fisher Scientific (2 mentions)
Pdonr223 pi4kap2, by Addgene (2 mentions)
Shrna vector, by OriGene (2 mentions)
Mnd gfp cassette, by OriGene (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
S3e cell sorter, by Bio-Rad (1 mentions)
Powerup sybr green master mix kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 thermocycler, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Phusion proofreading polymerase, by Thermo Fisher Scientific (1 mentions)
Revertaid cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Neon electroporation system, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
10 protocols using clonejet kit
1
SNCG Gene Isoform 2 Cloning and Expression
2
CRISPR Mutation Screening by T7EI Assay
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Cells were seeded at the amount of 1 cell per well in a 96-well plate by FACS (S3e Cell Sorter, Bio-Rad) 48 h after transfection. After reaching a ∼80–90% confluency, cells were divided into two equal portions and seeded in two 96-well plates. One of the plates was used for mutation screening by T7 endonuclease I (T7EI) cleavage assay (Kim et al., 2009 (link)). Genomic DNA was isolated using genomic DNA Isolation Kit (BIOLABMIX Ltd., Novosibirsk, Russia), PCR was performed using specific primers (5’-AGCCTTTGTCTGCTAAGGTCA-3’ and 5’-GTTGCCATTAACCGATGTCGA-3’ for SNORD74, 5’-TGGTATGTTACCTGCATCATTGG-3’ and 5’-TAGGTGTACTCTCTATGTTCCC-3’ for SNORD75, 5’-GAGTGCTAGAATGATGAGG-3’ and 5’-TCCAGCTTTCTGTCTAATGCC-3’ for SNORD77, 5’-ATTACAGGCATGTGACACC-3’ and 5’-CACTCCCATCTACAGATTAAGG-3’ for SNORD80), and the amplification products were annealed and subjected to T7EI (NEB) according to the manufacturer’s protocol. Mutations were identified by TA-cloning of the PCR products using CloneJET Kit (Thermo Fisher Scientific) and E. coli strain XL1-Blue followed by Sanger sequencing with further analysis of the obtained data by Tracking of Indels by Decomposition (TIDE) assay (Brinkman et al., 2014 (link)).
3
Identification and Characterization of CTX-M Genes
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UTI isolates were sub-cultured on primary UTI Agar (#2421517) at 37 °C for 18–24 h to identify Enterococcus spp., Escherichia coli and coliform bacteria. DNA was isolated, and the CTX-M genes were amplified as described in [6 (link)]. Primers: CTX-M group 1: F: 5′-TTAGGAARTGTGCCGCTGYA-3′, R: 5′-CGATATCGTTGGTGGTRCCAT-3; CTX-M group 2: F: 5′-CGTTAACGGCACGATGAC-3, R: 5′-CGATATCGTTGGTGGTRCCAT-3; CTX-M group 9: F: 5′-TCAAGCCTGCCGATCTGGT-3, R: 5′-TGATTCTCGCCGCTGAAG-3; and CTX-M group 8/25: F: 5′AACRCRCAGACGCTCTAC-3, R: 5′-TCGAGCCGGAASGTGTYA3T. DNA fragments were purified and sequenced after cloning into the pJET1.2 plasmid (CloneJET kit/ThermoFischer Scientific/(K1231)).
4
In Vitro Transcription of Viral RNA
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Positive control RNA for RT-qPCR assays was produced via in vitro transcription (IVT) from pDNA templates, obtained by cloning the corresponding viral cDNA fragments to pJet using CloneJet Kit (Thermo Scientific, Waltham, USA). Bacterial clones were grown in LB media with ampicillin 100 μg/mL and plasmid DNA was extracted with Evrogen (Russia) plasmid midi kit.
5
Absolute Quantification of ZIKV and CHIKV Genomic Copies
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For the absolute quantification of the genomic copies, 76 pb of E and 125 pb of NSP4 of ZIKV/Col and CHIKV/Col were cloned, respectively [52 (link)]. The amplified PCR products were ligated into the pJET1.2/blunt vector and cloned using the CloneJET kit (Thermo Scientific, Waltham, MA, USA), following the previously described protocol [58 (link),59 (link)]. Total viral RNA extraction from the cell monolayers was conducted with the ZYMO® Quick-RNA™ Viral kit, retrotranscription (from 500–1000 ng of RNA) with the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific®), and amplification with the Power Up™ SYBR Green Master Mix kit (Thermo Scientific®), following the manufacturers’ instructions. The samples were amplified in a QuantStudio 3 thermocycler (Thermo Scientific®); the thermal profile used was 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 2 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
6
Cloning and Ligation of Pi4kap2 into pLV Vector
7
Cloning Brassica napus Gene into Arabidopsis
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Genomic DNA from Brassica napus was amplified using Phusion Proofreading polymerase (Thermo Fischer) and primers with specific restriction sites. The amplified DNA was separated on an agarose gel and extracted using a GeneJet gel extraction kit (Thermo Fischer) and then ligated into a pJET2.1 cloning vector using the CloneJet kit (Thermo Fischer). The insert was digested and separated on an agarose gel and then cloned into a pHPT1 binary vector [44 (link)], using T4 Ligase (Thermo Fischer). The resulting construct was transformed into Agrobacterium and then into Arabidopsis srr1–1 plants using the floral dip method.
8
Cloning and Ligation of Pi4kap2 into pLV Vector
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The Pi4kap2 coding sequence was amplified from pDONR223-PI4KAP2 (23601 Addgene) using primers (Supplemental Experimental Procedures ) that introduced an HA-tag at the 3′ end and flanked the fragment with PstI and AgeI restriction enzyme sequences. The fragment was ligated into the pJet cloning vector using CloneJET kit instructions (K1231 Thermo Fisher Scientific) and amplified in Stbl3 bacteria (C737303 Thermo Fisher Scientific). The pLV-Ef1a-MCS-IRES-RFP-puro plasmid and the Pi4kap2HA-pJet vector were linearized with PstI and AgeI. The Pi4kap2-HA fragment was gel purified and ligated into the pLV vector using T4 ligase. For the shRNA vector purchased from Origene (TL510615), the MND-GFP cassette (a gift from the Kohn Laboratory) was used to replace the CMV-GFP reporter.
9
Zebrafish 12-LOX Expression Plasmid Construction
10
TP53 Knockout in RPE1 Cells
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RPE1 TP53−/− and TP53+/+ cells are generated by electroporation of 106 cells with 10 µg gRNA inserted pX458 with Neon electroporation system (#MPK10025; Thermo Fisher Scientific) according to the manufacturer’s protocol. 48 h after electroporation, GFP-positive cells are single-sorted in 96-well plates containing full DMEMF12 medium. When cells are grown enough, 96-well plates are duplicated, and one plate was used for genomic DNA isolation by QuickExtraction DNA isolation solution (#QE09050; Epicentre). The cells with a negative WT band (obtained only in RPE1 TP53−/− background) were cultured further for mRNA extraction (RNeasy Plus Mini Kit, #74134; Qiagen). The resulting mRNA was converted to cDNA by RevertAid cDNA synthesis kit (#K1691; Thermo Fisher Scientific) according to the manufacturer’s protocol. 2 µl of produced cDNA (out of 20 µl) was used in PCR reactions to amplify with forward (5′-ATCCAAGGAACTCTCAAAGCAAGGATACTGT-3′) and reverse (5′-ATGAACGAGACGATGCAGCAGACTG-3′). PCR products were cloned into pJET2.1 (CloneJET Kit; #K1231; Thermo Fisher Scientific) for sequencing with pJET1.2 forward and reverse primers. Conserved sequences were identified by Clustal omega tool of European Molecular Biology Laboratory (Sievers et al., 2011 (link)) and aligned by Jalview tool (Waterhouse et al., 2009 (link)).
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