S. equi isolates, which differed in their subspecies identification on the automated biochemical platform and the MALDI-TOF MS system, were subsequently tested by PCR following a previously published protocol 2 with minor modifications. Total DNA was extracted from bacterial colonies using the DNeasy blood and tissue kit (Qiagen, Valencia, CA), following the manufacturer's recommended protocol. For each S. equi isolate, 2 separate real-time PCR reactions were performed targeting either the sodA gene or seeI gene. 2 Both PCRs were performed in a 25-µL final reaction volume. The sodA PCR reaction mix contained 0.6 µM each of the forward and reverse primers (Integrated DNA Technologies, Coralville, IA), 0.2 µM probe (Promega, Madison, WI), and 2 µL of template DNA. The seeI PCR reaction mix contained 0.9 µM each of the forward and reverse primers (Integrated DNA Technologies), 0.2 µM hydrolysis probe (Promega), and 2 µL of template DNA. For both PCR assays, 1× buffer (Promega) and 0.5 µL of Taq DNA polymerase (Promega) were used. The thermocycling conditions were set at 95°C for 8 min, followed by 45 cycles of 95°C for 20 s, and 60°C for 60 s. The PCR was performed on a real-time PCR system (Applied Biosystems, Thermo Scientific, Foster City, CA).
Forward and reverse primer
Manufactured by Integrated DNA Technologies
Sourced in United States, Belgium, United Kingdom, Japan
Forward and reverse primers are short synthetic DNA sequences that are used to initiate the amplification of a specific target DNA sequence during a polymerase chain reaction (PCR) process. They serve as the starting points for DNA synthesis, enabling the targeted replication of a genetic region of interest.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy kit, by Qiagen (3 mentions)
Powertrack sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (2 mentions)
Sybr select master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Phusion polymerase, by Thermo Fisher Scientific (2 mentions)
Stratagene mx3005p, by Agilent Technologies (2 mentions)
Sybr green master mix, by Roche (2 mentions)
Nuclease free water, by Roche (2 mentions)
Lightcycler 96 real time pcr machine, by Roche (2 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Lightcycler 480 system, by Roche (2 mentions)
Lightcycler 480 sybr green 1 master enzyme mix, by Roche (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pcr master mix, by Promega (2 mentions)
55 protocols using forward and reverse primer
1
PCR-based Subspecies Identification of S. equi
2
Site-Directed Mutagenesis of Lysine to Arginine
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Mutagenesis of lysine to arginine residues carried out by first designing forward and reverse primers (Integrated DNA Technologies) containing the desired point mutation. PCR was performed using a Phusion High-Fidelity DNA Polymerase (New England BioLabs) per manufacturer’s protocol. PCR products were digested with DpnI enzyme (NEB) at 37 °C for 2 h prior to transformation. Constructs were validated by sequencing.
3
Site-directed mutagenesis of EsaR variants
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Sequencing revealed that the genes encoding several EsaR* variants had multiple mutations (Table 2 ). Consequently each individual mutation was separately regenerated via site-directed mutagenesis. Forward and reverse primers containing the desired nucleotide substitutions were designed (Integrated DNA Technologies) and used in PCR reactions in conjunction with upstream vector primer BADVF or downstream vector primer BADR or BADR500 (Table S1 ). Following the first round of PCR, the desired fragment was gel purified using a Qiaquick Gel Extraction Kit (Qiagen) and a second round of PCR with external primers BADVF and BADR or BADR500 was performed in order to obtain a full length esaR gene containing the mutation. Upon completion of the second round of PCR, the full-length esaR* gene and pBAD22 vector were digested with NheI and HindIII (NEB). The resulting fragments were ligated together and transformed into E. coli Top10 pRNP-lacZ. Blue/white screening and β-galactosidase assays were used to determine EsaR* variants unable to respond to AHL. Plasmids conferring ampicillin resistance were isolated and sequenced (VBI).
4
Glioblastoma Organoid RNA Extraction
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Live cultured glioblastoma organoids underwent one cycle of staining and quenching. After 24 h post cyclic imaging, RNA was extracted from the organoids with Trizol Reagent (ThermoFisher; 15596018), labeled with GlycoBlue Coprecipitant (ThermoFisher; AM9515), and treated with 2U of TURBO DNase (ThermoFisher; AM1907) at 37 °C for 30 min. DNase in activation Reagent was then added to inactivate the TURBO DNase at 0.1 volume for 5 min at RT. The iScript cDNA Synthesis Kit (BioRAD; 1708890) was used for RT‐PCR. With this kit, the cell RNA was mixed with its reagents and run in a SimpliAmp Thermal Cycler (Applied Biosystems) as per the manufacturer's protocol. The qPCR master mix includes PowerTrack SYBR Green Master Mix (Applied Biosystems; A46109), nuclease‐free water, and forward and reverse primers (Integrated DNA Technologies). 10 µL of the qPCR master mix was added to each well. For every qPCR experiment, 40 cycles were run using a QuantStudio 3 Real‐ Time PCR machine (Applied Biosystems). Organoid gene assessment was derived from patients UP‐9121.
5
Cloning and Expression of Crm1 and Ran
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All plasmids used in this study are listed in Supplementary file 1 . Crm1 and Ran were cloned between the NdeI and XhoI sites in a pET19b vector (EMD Millipore, Darmstadt, Germany) that encodes an amino-terminal deca-histidine tag followed by a tobacco etch virus (TEV) protease site. Genes were amplified from their source vectors by polymerase chain reaction with phusion polymerase (New England Biolabs (NEB), Ipswich, MA) using forward and reverse primers (Integrated DNA technologies (IDT), Coralville, IA, listed in Supplementary file 2 ) containing NdeI or XhoI restriction sites, respectively. PCR products and the target vector were digested with restriction enzymes (NEB), purified (Qiagen, Venlo, Netherlands), ligated (Roche, Basel, Switzerland), and then transformed into E. coli DH5α. Plasmids from positive transformants were isolated (Qiagen), screened by restriction digests, and sequenced (University of California - Berkeley DNA Sequencing Facility, Berkeley, CA).
6
Quantitative Real-Time PCR Analysis
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The total RNA was prepared using an Ambion® RiboPureTM Kit (Thermo Fisher Scientific, Waltham, MA, USA), and 100 to 500 ng samples were reverse-transcribed into cDNA using PrimerScriptTM RT Master Mix (Takara Bio USA, Inc., San Jose, CA, USA). Next, 6–15 ng samples of cDNA were used for the detection of each gene. The qRT-PCR process was performed on a CFX384 TouchTM Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using SYBRTM Select Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) with 100 nM of the forward and reverse primers (Integrated DNA Technologies, Coralville, IA, USA) shown in Table 1 . The data were analyzed using Bio-Rad CFX Maestro software. A relative quantification analysis was performed following the 2−ΔΔCT method, and the data were normalized to S18 expression.
7
Quantitative Gene Expression Analysis
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RNA extraction was performed, practicing the manufacturer’s instructions of miRNeasy kit (QIAGEN) including on-column deoxyribonuclease treatment, and NanoDrop (Thermo Fisher Scientific) was used to determine the concentration. Complementary DNA (cDNA) synthesis was achieved, practicing the iScript Advanced cDNA synthesis kit instructions from Bio-Rad. The PCR mix contained 5 ng of cDNA, 2.5 μl of SsoAdvanced SYBR qPCR super mix (Bio-Rad), and 0.25 μl of forward and reverse primers (to a final concentration of 250 nM; Integrated DNA Technologies). The RT-qPCR cycling analysis was performed using a LC-480 device (Roche). qBasePlus software 3.2 (www.biogazelle.com ) was used for the analysis of the gene expression levels. For the neuroblastoma cell lines, the following reference genes were used: B2M, HPRT1, TBP, and YHWAZ. The error bars in figures represent SD after error propagation, with mean centering and scaling to control. The primer designs are provided on table S6.
8
SFB Gene Amplification Protocol
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SFB gene amplification was performed starting from 150 ng of genomic DNA, in a final volume of 30 µL. Amplification was carried out in the presence of 1X buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100), 33 µM dNTPs, 0.5 µM forward and reverse primers (IDT Integrated DNA Technologies, Coralville, Iowa, USA), 2U of DyNAzyme II DNA Polymerase (Finnzymes, Espoo, Finland) and ultrapure water. Primers features are summarised in table 1 . Each reaction was subjected to an initial denaturation step of 3 min at 96°C. The 40 PCR cycles used were as follows: denaturation, 15 s at 95°C; annealing, 30 s at 58°C; elongation, 20 s at 72°C. The amplification was performed using Gene Amp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, California, USA). PCR products were analysed by 2.5% agarose-gel electrophoresis, using ChemiDoc MP System (Bio-Rad), image acquisition and analysis was performed using Image Lab Software V.4.0 (Bio-Rad). The molecular weight marker used was GeneRuler 50 bp DNA Ladder (Thermo Scientific) designed to sizing a large wide range of double-stranded DNA.
9
Quantitative Real-Time PCR Analysis
10
Plasmid-based HIV-1 MA Protein Expression
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A plasmid encoding for HIV-1 MA (pNL4-3 strain) and yeast N-terminal myristoyl transferase was provided by Dr Michael Summers (Howard Hughes Medical Institute, University of Maryland). The A45E, T70R, Q63R and L75G MA mutant constructs were generated using a QuickChange Lightning site-directed mutagenesis kit (Agilent Technologies). Forward and reverse primers (Integrated DNA Technologies) extended 15 base pairs on either side of the mutation codon. Mutations were verified by plasmid sequencing at the Heflin Genomics Core at the University of Alabama at Birmingham. WT and mutant MA and myr(–)MA proteins were prepared as described (22 (link), 24 (link), 25 (link), 26 (link)).
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