The quantification of A. apis was checked by qPCR analysis among three different sources: all the contaminated pollen batch samples with the fungus (n = 6), infected living larvae (n = 9) and symptomatic dead larvae (n = 3). Ascosphaera apis quantification was performed by using a CFX96 Real-Time PCR Detection system (Bio-Rad, Hercules, CA, USA), performing each reaction in triplicate. The total reaction volume of 20 µL contained 10 µL of GoTaqr qPCR Master Mix, (Promega, Madison, WI, USA), 1 µL (10 µM) Forward and 1 µL (10 µM) Reverse primer targeting the 18S rRNA gene (see Supplementary Table 1 ) and 8 µL of diluted DNA. Nuclease free water was used as a negative control (NTC). The standard curve was obtained from the highest infected sampled and was serially diluted (E = 93.2%; R2 = 0.995).
Gotaq r qpcr master mix
Manufactured by Promega
Sourced in United States
GoTaq(R) qPCR Master Mix is a ready-to-use solution for quantitative polymerase chain reaction (qPCR) experiments. The master mix contains all the necessary components, including a DNA polymerase, for performing qPCR.
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Lab products found in correlation
Superscript 2 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Trizol rna isolation reagent, by Thermo Fisher Scientific (3 mentions)
Tri reagent, by Merck Group (2 mentions)
Superscript vilo kit, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
Rnaseout ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Lineage cell depletion kit for mice, by Miltenyi Biotec (1 mentions)
Anti sca 1, by Miltenyi Biotec (1 mentions)
Anti gr1, by Miltenyi Biotec (1 mentions)
Anti ter 119, by Miltenyi Biotec (1 mentions)
Ab 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Primerdesign (1 mentions)
7500ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
C1000 touch, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
14 protocols using gotaq r qpcr master mix
1
Quantification of Ascosphaera apis in Honey Bee Samples
2
Quantification of PERK Silencing by RT-qPCR
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Total RNA content of cells was extracted using TRIzol RNA isolation reagent (Invitrogen) [41 (link)]. Retrotranscription was performed using SuperScriptII First-Strand Synthesis System (Invitrogen). Nucleic acid levels were measured using NanoDrop2000 UV calculator. Equal amounts of cDNA were used for real-time PCR to check the efficiency of PERK silencing. PCR reaction and real-time detection was performed using GoTaq(R) qPCR Master Mix (Promega, Madison, WI, USA, A6002) and STRATAGENE Mx3005P Real-Time PCR Detection System. The real-time PCR thermocycles were the followings: 95 °C 10 min (1×), 95 °C 30 s, 58 °C 45 s, 72 °C 30 s (40×), 95 °C 5 min, 55 °C 1 min, 97 °C 30 s (1×). The appropriate forward and reverse real-time PCR primers were used for PERK and GAPDH.
3
Murine Hematopoietic Cell Analysis
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TRI reagent was from Sigma-Aldrich (Madrid, Spain). Antibodies for the detection of murine CD135 (Flt3), CD34, CD16/32, and CD127 (IL-7Rα) were from eBioscience (Barcelona, Spain). The Lineage Cell Depletion Kit for mice, FcR Blocking reagent and murine flow cytometry antibodies (CD3e, CD4, CD8a, CD11b, CD19, CD43, CD45R (B220), CD117 (c-Kit), anti-Gr1, anti-Sca-1, anti-Ter-119, anti-IgM) were from Miltenyi Biotec (Madrid, Spain). Super Script II reverse transcriptase and RNase OUT ribonuclease inhibitor were from Invitrogen and Thermo Fisher Scientific (Madrid, Spain). GoTaqR qPCR master mix was from Promega (Madrid, Spain).
4
Measuring Gadd34 Expression via qRT-PCR
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Total RNA content of cells was extracted using TRIzol RNA isolation reagent (Invitrogen) [34 (link)]. Retrotranscription was performed using SuperScriptII First-Strand Synthesis System (Invitrogen). Nucleic acid levels were measured using GenQuant pro RNA/DNA calculator. Equal amounts of cDNA were used for real-time PCR to check the efficiency of Gadd34 silencing. PCR reaction and real-time detection was performed using GoTaq(R) qPCR Master Mix (Promega, A6002) and STRATAGENE Mx3005P Real-Time PCR Detection System. The real-time PCR thermocycles were the followings: 95°C 10 min (1x), 95°C 30 sec, 58°C 45 sec, 72°C 30 sec, (40x), 95°C 5 min, 55°C 1 min, 97°C 30 sec (1x). The appropriate forward and reverse real-time PCR primers were used for Gadd34 and GAPDH.
5
Equine Stem Cell Gene Expression Analysis
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RNA was extracted with Trizol and using a cell scraper for cell detachment and by repeated pipetting to disrupt cell pellets. TSPCs were analyzed at passage 1–2. cDNA was synthesized in a 25 μl reaction from 1–2 μg of total RNA by incubation for 5 min at 70°C, 60 min at 37°C, and 5 min at 93°C using M‐MLV reverse transcriptase and random‐hexamer oligonucleotides (Promega Ltd., Southampton, UK). qRT‐PCR was conducted using a GoTaq(R) qPCR Master Mix (Promega). A total of 10 ng of cDNA was amplified in a 25 μl reaction using an AB 7300 Real Time PCR System (Life Technologies Ltd., Paisley, UK). Equine specific gene‐specific primers were used (Table 1 ) and GAPDH or B2M (Primer Design, Southampton, UK, proprietary sequence) used as internal controls. After an initial denaturation for 10 min at 95°C, 40 PCR cycles were performed consisting of 15 s at 95°C and 1 min at 60°C. Relative gene expression was calculated according to the comparative Ct method.23
6
Quantitative Analysis of GADD34 Gene Expression
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Total RNA content of cells was extracted using TRIzol RNA isolation reagent (Invitrogen) [33 (link)]. Retrotranscription was performed using SuperScriptII First-Strand Synthesis System (Invitrogen). Nucleic acid levels were measured using GenQuant pro RNA/DNA calculator. Equal amounts of cDNA were used for real-time PCR to check the efficiency of GADD34 silencing. PCR reaction and real-time detection were performed using GoTaq(R) qPCR Master Mix (Promega, A6002) and STRATAGENE Mx3005P Real-Time PCR Detection System. The real-time PCR thermocycles were the following: 95°C 10 min (1x), 95°C 30 sec, 58°C 45 sec, 72°C 30 sec (40x), 95°C 5 min, 55°C 1 min, and 97°C 30 sec (1x). The appropriate forward and reverse real-time PCR primers were used for GADD34 and GAPDH.
7
Gene Expression Quantification by qPCR
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Total RNA was isolated from cultured cells using the NucleoSpin RNA (22740955.250) according to manufacturer’s instructions. Samples were treated with DNase I before reverse transcription. cDNA was generated from 1 μg RNA using random hexamers and reverse transcriptase (TaqMan Reverse Transcription Reagents N8080234). qPCR amplification and analysis were conducted using the 7500HT Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA) using the GoTaq(R) qPCR Master Mix (Promega, Madison, WI, USA). RNA levels were normalized to HPRT or GAPDH expression using the DDCt method. Primer sequences are provided in Supplementary Table S1 .
8
Quantitative RT-PCR Analysis of Mouse Kidney
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Total RNA was extracted from fresh mouse kidneys of each group using the TRIzol method, with samples having no DNAse/RNAse activity. Then, a 1 µg sample of RNA was reverse-transcribed into cDNA using the GoScript Reverse Transcription System, according to the manufacturer’s protocol (Promega). The qRT-PCR was performed on an ABI7500 system using the GoTaqR qPCR Master Mix (Promega). All data were normalized to the expression of β-actin, and relative RNA expression was calculated using the 2−δδCT method. All primer sequences are in Supplementary Table 1
9
Quantitative Analysis of Focal Adhesion Proteins
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Total RNA was isolated using the NucleoSpin RNA II kit (MACHEREY-NAGELl) according to the manufacturer’s instructions. 1 µg RNA was reverse-transcribed using the SuperScript VILO kit (Thermo Fisher Scientific). PCR amplification of the cDNA from the reverse transcription reaction was performed using specific primer pairs for mouse paxillin (forward: 5′-GAGCAGTCCGCAGCGAGTCA-3′; reverse: 5′-ACGGCCGCTCTCCATCCACTC-3′), mouse Hic-5 (forward: 5′-CGATGTGGCTTCTGTAACCAAC-3′; reverse: 5′-ACCCTCTTCTCCAAAAGGCTC-3′), and mouse leupaxin (forward: 5′-TGCCTCCCAAAACCTCAGCAGC-3′; reverse: 5′-TCCTGCTGGTCTGGCAAGGGT-3′). Quantitative real-time PCR was performed with GoTaqR QPCR Master Mix (Promega) in a 25-µl reaction on a thermal cycler (C-1000 Touch; Bio-Rad Laboratories). Ct values were determined with the same software, and normalization was conducted with housekeeping genes actin, RanBP1, GAPDH, and ATP50, which yielded similar results. The expression levels of each target gene in the siRNA-treated cells were calculated with GAPDH as a reference gene and compared with the controls.
10
Quantitative RT-PCR Analysis of ASC/TMS1 Gene
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Total RNA was extracted from RCC cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA). For semiquantitative RT-PCR, ASC/TMS1 gene was amplified using AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) as reported previously. Real-time PCR was performed using GoTaq(R) qPCR Master Mix (Promega Biotech, Madison, WI, USA) according to the manufacturer's protocol on 7500 Fast Real-Time PCR System (ABI). Primer sequences and PCR conditions are listed in Table 2 . GAPDH was used as the housekeeping gene for loading control.
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