Plasmids were linearized using the respective restriction enzyme (
Rneasy minelute rna cleanup kit
Manufactured by Qiagen
Sourced in United States
The RNeasy MinElute RNA Cleanup kit is a laboratory tool designed to purify and concentrate RNA samples. It utilizes a silica-membrane technology to efficiently remove contaminants and impurities, allowing for the recovery of high-quality RNA suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 3130xl genetic analyzer, by Hitachi (2 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Midiprep kit, by Qiagen (1 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (1 mentions)
Ambion mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions)
Beacon designer software, by Premier Biosoft (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Rnase free dnase 1, by Roche (1 mentions)
Dna miniprep kit, by Qiagen (1 mentions)
T7 mmachine kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using rneasy minelute rna cleanup kit
1
Plasmid Preparation and cRNA Synthesis for Aquaporin Expression
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Table 2 contains a list of the expression vectors, restriction enzymes and promoters used in this study. The plasmids encoding for hAQP1, hAQP1FLAG, hAQP1C189S mutant, rAQP3, hAQP7, hAQP8 or rAQP9 were transformed into TOP10 competent cells. All of the plasmids were sequenced using the BigDye kit and the ABI Prism 3130XL Genetic Analyzer (Hitachi, Tokyo, Japan). Plasmid DNA was purified using either miniprep or midiprep kits (Qiagen). DNA concentration and purity were determined spectrophotometrically on a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).
Plasmids were linearized using the respective restriction enzyme (Table 2 ) and restriction-digested products were purified using the QIAquick PCR purification kit (Qiagen). The capped RNAs (cRNAs) were transcribed using either a T3 or T7 mMessage mMachine kit (Ambion, Austin, USA) depending on the promoter present on the linearized DNA (Table 2 ). The cRNA was then purified and concentrated using the RNeasy MinElute RNA Cleanup kit (Qiagen) and quantified by measuring the absorbance at 260 nm using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific).
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Plasmids were linearized using the respective restriction enzyme (
2
SHAPE Analysis of RNA with LNA Probes
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For structure verification experiments, a truncated variant of AON 445569 (Wheeler et al. 2012 (link)), LNA oligonucleotide tr445569, 5′-G TTG TG AACTG -3′, or LNA (CAG)3C, 5′-C AGC AG CAGC-3′ (LNA nucleotides depicted in bold, Exiqon), was added before the denaturation step in a 1:10 RNA:AON molar ratio. SHAPE was performed as described above, only with an additional purification step after the modification because the bound LNAs interfered with the RT reaction (data not shown). Inspired by Busan et al. (Busan and Weeks 2013 (link)), after recovering the modified RNA, a 50-fold excess of DNA AON (5′-CAGTTCACAAC-3′ or 5′-GCTGCTGCTG-3′; Exiqon) was added and then heated at 80°C for 5 min. After placing on ice, the RNA was directly purified from LNAs and oligos using the RNeasy MinElute RNA Cleanup kit (QIAgen). RT reactions with primer D14 (in the case of LNA tr445569) or primers D14, D16, and D2 [for LNA (CAG)3C] and data analysis were performed as described above. Because a slightly altered purification method was used compared to SHAPE without LNA perturbation, additional reactions without LNAs were performed in parallel.
3
Construct RNA Injection Protocol
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Constructs were cloned into pCSDest (Villefranc et al., 2007 (link)), linearized, transcribed using the Ambion mMessage mMachine SP6 Kit (Thermo Fisher Scientific), and purified with the RNeasy MinElute RNA Cleanup Kit (Qiagen). RNA was quantified based on absorbance at 260 nm and injected with 0.1% phenol red into 1–2 cell embryos in a volume of 1–2 nl.
4
RNA Isolation and qPCR Analysis
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Total RNA isolation was performed as previously described with minor modifications (Vogelaar et al. 2009 (link)). Briefly, after washing the cultures with PBS, explants and adhering axons were collected in lysis buffer, pooling 2–3 wells per sample. After RNA isolation using RNeasy Micro Kit (Qiagen) according to manufacturer’s instruction, samples were subjected to RNase-free DNaseI (Roche) and further purified using the RNeasy MinElute RNA cleanup kit (QIAGEN). cDNA synthesis was performed with the Superscript III First-Strand Synthesis System (Invitrogen) using a mix of random primers and Oligo(dT). Primers (Suppl. Table S1) were designed using the Beacon Designer Software (Premier Biosoft) and optimized on control brain cDNA to determine optimal primer concentration, annealing temperature and efficiency. qPCRs were run in 96-wells plates in the CFX96™ Real-Time PCR Detection System (Biorad). Levels of target genes were calculated in relation to housekeeping genes as previously described (Vogelaar et al. 2009 (link)).
5
Molecular Cloning and Characterization of Murine Urea Transporters
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The UT-A3 (AF258602), UT-A2 (AF367359) and UT-B (AF448798) were a gift from Dr Bryce MacIver, Harvard Medical School, MA, USA. DNA sequences encoding C-terminally c-Myc tagged murine UTs: mUT-B, mUT-A2 and mUT-A3 were subcloned into the P7TS expression vector. The resulting plasmids were transformed into TOP10 competent cells via heat shock, and purified with a DNA Miniprep kit (part #28104, Qiagen, Valencia, CA, USA). All of the plasmids were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (part #4337455, Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3130XL Genetic Analyzer (HITACHI, Tokyo, Japan).
All UT-encoding cDNAs were linearised with XbaI restriction enzyme (part #R0145S, New England Biolabs, Ipswich, MA, USA) and purified using the QIAquick PCR Purification Kit (part #27106, Qiagen). The linearised and purified DNAs were transcribed into capped RNA (cRNA) using the T7 mMachine Kit (part #AM1344, Ambion, Austin, TX, USA) and the cRNAs were purified with the RNeasy MinElute RNA Cleanup Kit (part #74204, Qiagen). The concentration and purity of all DNAs and RNAs were quantified using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
All UT-encoding cDNAs were linearised with XbaI restriction enzyme (part #R0145S, New England Biolabs, Ipswich, MA, USA) and purified using the QIAquick PCR Purification Kit (part #27106, Qiagen). The linearised and purified DNAs were transcribed into capped RNA (cRNA) using the T7 mMachine Kit (part #AM1344, Ambion, Austin, TX, USA) and the cRNAs were purified with the RNeasy MinElute RNA Cleanup Kit (part #74204, Qiagen). The concentration and purity of all DNAs and RNAs were quantified using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
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