WAVE3 constructs were generated using the PCR from the template WAVE3 cDNA IMAGE clone 4838122 (ATTC) as described previously [19 (link)]. Briefly, the ATG sequence for the first methionine was replaced by a TTG. Amplified fragments were subcloned into the pCR2·1 vector using the T-A cloning kit (Invitrogen). The subcloned fragments were inserted into EcoRI-digested pEGFPC2 vector (Clontech), in-frame with the 3′ terminus of EGFP (GFP). All constructs were sequence-verified using a 3100 Genetic Analyzer (ABI Prism). The EGFP-recombinant vectors were used for either transient or stable transfections using standard protocols, and the correct size of the fusion proteins were verified by western blot analysis. Oligonucleotide primers used for PCR and cloning were from SABiosciences and are available upon request.
Oligonucleotide primers
Manufactured by Qiagen
Sourced in Singapore
Oligonucleotide primers are short, synthetic DNA sequences used in various molecular biology techniques, such as polymerase chain reaction (PCR) and sequencing. They serve as the starting point for DNA synthesis and amplification, providing the necessary complementary sequences to initiate and guide the DNA replication process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Pegfp c2 expression vector, by Takara Bio (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system c1000 touch thermal cycler, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qiaamp dneasy blood and tissue kit, by Qiagen (1 mentions)
Qiaexpert, by Qiagen (1 mentions)
Prime taqtm dna polymerase, by GeNet Bio (1 mentions)
Twistamp, by Twist Bioscience (1 mentions)
100 bp dna ladder, by New England Biolabs (1 mentions)
8 protocols using oligonucleotide primers
1
Generation of WAVE3-EGFP Fusion Constructs
2
Quantitative RNA Expression Analysis
3
Extraction and Quantification of RNA
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Total RNA was extracted from cancer cell lines or tumor tissue using TRIzol reagent (Invitrogen), following the manufacturer's instructions. cDNA was generated and used as a template for qRT-PCR as described previously [68 (link)]. Oligonucleotide primers used for qRT-PCR were from SABiosciences (Supplementary Table 1 ).
4
NMDA Receptor Subunit Expression Analysis
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Amplification of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in real-time polymerase chain reaction (PCR) was performed with oligonucleotide primers purchased from Qiagen company primer bank. Normalization of target genes expression was achieved using the beta-actin as the housekeeping gene where its primers were also ordered from Qiagen company.
5
GFP-tagged WAVE3 PRD Construct Generation
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GFP-tagged WAVE3 PRD constructs were generated as previously described14 (link) and sequence-verified. GFP-tagged WAVE3 PRD was generated via ligation of the wild-type WAVE3 PRD domain (accession number AF454702) in- frame with GFP in the pEGFP-C2 expression vector (Clontech). The resulting plasmid construct was used as a template for site-directed mutagenesis to introduce the Y to F mutations using the primers listed in Table 1 as described previously16 (link). The GFP-recombinant vector or the empty GFP expression control vector was used for stable transfections using standard protocols. The correct size of the fusion proteins was verified by Western blot analysis. Oligonucleotide primers used for site-directed mutagenesis and sequencing were from Qiagen and are described in Table 1 and reference16 (link).
6
Quantitative RT-PCR Analysis of Gene Expression
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RNA extraction was performed as previously described26 (link). Briefly, cells were lysed in TRIzol reagent (Invitrogen), and total RNA was extracted according to the manufacturer’s instructions. Total RNA, resuspended in RNase-free water, was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific); and 1 μg of RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis System RT-PCR kit (Invitrogen). The resulting cDNA was used a template for qtRT-PCR as described previously13 (link), using the C1000 Touch Thermal Cycler CFX96 Real-Time System (Bio-Rad). Oligonucleotide primers used for qtRT-PCR were from Qiagen and are listed in Table 1 .
7
Generation of GFP-tagged WAVE3 Constructs
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GFP-tagged WAVE3 constructs were generated as previously described36 (link). The GFP-recombinant vector or the empty GFP expression control vector was used for stable transfections using standard protocols34 (link). Oligonucleotide primers used for site-directed mutagenesis and sequencing were from Qiagen, were previously reported36 (link), and are available upon request.
8
Leptospirosis Detection in Canine and Wildlife
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Reference culture, collection of samples, and biological reagents A panel of Leptospira reference strains to represent twenty-four serogroups (23 pathogenic and one nonpathogenic strain) maintained at the Zoonoses Research Laboratory (ZRL), Tamil Nadu Veterinary and Animal Sciences University, Chennai was used as the source of positive control DNA (Table 1). The clinical samples included 150 serum samples from dogs with clinical signs of fever, jaundice, vomition, hematuria, renal failure submitted to ZRL for Leptospira diagnosis (from Madras Veterinary College Teaching Veterinary Clinical Complex and Veterinary University Peripheral Hospital of TANUVAS and some Private Veterinary Clinics from Chennai). The kidney tissues of wild rats (n=28) and water samples (n=15) collected from selected locations in and around Chennai during the monsoon rain (during December 2020) were also used in this study. The other materials used in the study included Prime TaqTM DNA polymerase (GeNet Bio, Korea), TwistAmp® (TwistDx, UK), QIAamp DNeasy Blood and Tissue Kit, QIAamp DNA Mini Kit (Qiagen, India), 100bp DNA ladder (New England Biolabs, MA), Oligonucleotide primers (synthesized from IDT, Singapore) and QIAexpert (Qiagen, MA). The volumes dispersed by the different micropipettes used in the study were also veri ed at different time points as part of the validation process.
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