Total RNA was extract from human fibroblast (C-013-5C, Life Technologies), human iPS cells (SBI) and hDSCs by the single-step method of isolation using Trizol (Invitrogen), and then was reverse transcribed with random hexanucleotides using the SuperScript III First-Strand Synthesis System (Invitrogen) as prescribed described69 (link). PCR was performed with JumpStart Taq DNA polymerase (Sigma). In quantitative PCR, 50 ng of total RNA was typically used as template in 20 ml SYBR green PCR reactions (40 cycles of 15 s, 95 °C/60 s, 60 °C) on Applied Biosystems 7300 that additionally contained 0.375 mM of each primer70 (link)71 (link) and 10 ml of SYBR green PCR mix (ABI).
Jumpstart taq dna polymerase
Manufactured by Merck Group
Sourced in United States, Germany
JumpStart Taq DNA polymerase is a thermostable DNA polymerase enzyme used in polymerase chain reaction (PCR) applications. It catalyzes the synthesis of DNA strands from nucleotide precursors.
Automatically generated - may contain errors
Lab products found in correlation
Lookout mycoplasma pcr detection kit, by Merck Group (10 mentions)
Random hexanucleotide primers, by Thermo Fisher Scientific (8 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Sybr green, by Thermo Fisher Scientific (6 mentions)
Minelute 96 uf pcr purification kit, by Qiagen (5 mentions)
Icycler iq5 thermal cycler, by Bio-Rad (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (3 mentions)
Sybr green 1, by Merck Group (3 mentions)
Superscript 2 rnase h reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Mj research ptc 200 thermal cycler, by Bio-Rad (3 mentions)
Powerplex 16 hs system, by Promega (3 mentions)
Pcr buffer, by Merck Group (3 mentions)
Sybr green pcr mix, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Dntps, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
76 protocols using jumpstart taq dna polymerase
1
Quantitative Transcriptional Analysis of Diverse Cell Types
2
Targeted miR-21 Knockout in HeLa Cells
3
Quantitative RT-PCR Analysis of CAIX
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Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) and the quantity of total RNA was measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA synthesis was performed using oligo (dT) 15 primer and GoScript Transcriptase according to manufacturer’s instructions (Promega GmbH, Mannheim, Germany). The PCR mixture contained: 1.5 uL of cDNA, 1× PCR buffer (Sigma Aldrich) 2.5 mM Magnesium Chloride (Sigma Aldrich), 0.2 mM dNTPs (Sigma Aldrich), 0.2 uM of each primer (Sigma Aldrich), and 1.25 U JumpStart™ Taq DNA Polymerase (Sigma Aldrich). The cDNA was amplified by PCR using the MJ Research PTC-200 Thermal Cycler with the following primers: CAIX (Carbonic Anhydrase 9) forward (5′-TACAGCTGAACTTCCGAGCG-3′), CAIX reverse (5′-CTAGGCTCCAGTCTCGGCTA-3′), HPRT1 (Hypoxanthine-Guanine Phosphoribosyltransferase) forward (5′-TGGCGTCGTGATTAGTGATG-3′), HPRT1 reverse (5′-TATCCAACACTTCGTGGGGT-3′). The PCR conditions for all analyzed genes were as following: denaturing at 95 °C for 5 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. The reaction was completed for 10 min at 72 °C. The PCR reaction was evaluated by checking the PCR products on 1.5% w/v agarose gels. Bands were normalized by use of HPRT1 to correct for differences in loading of the cDNAs.
4
Quantifying NLRP3 and IL-1β Expression
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Total RNA was extracted from 1 × 106 cells using TRIZOL® reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed with random hexamers (Roche, Indianapolis, IN, USA) using 2 μg RNA and Superscript II™ reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR was performed with the following primers: nlrp3-(F): 5′-ggagagacctttatgagaaagcaa-3′, nlpr3-(R) 5′-gctgtcttcctggcatatcaca -3′, IL1β-(F): 5′-ctgtcctgcgtgttgaaaga, IL-1β-(R) 5′ttgggtaatttttgggatctaca-3′, β-actin-(F) 5′-tatggagaagatttggcacc-3′ and β-actin-(R) 5′-gtccagacgcaggatggcat-3′, using JumpStart Taq DNA polymerase (Sigma, Saint Louis, MO, USA). PCR products were separated by electrophoresis on 1.8% agarose gel containing 0.02% ethidium bromide and visualized on a Scion Image gel imaging system. Subsequent quantification and analyses were performed using ImageQuant5.0 software (Molecular Dynamics).
5
Quantifying Colonic Gene Expression
6
LRRK2-IN-1 Impacts AsPC-1 Cell Transcription
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AsPC-1 cells (105/well) were seeded into 6-well plates in triplicate in the presence of LRRK2-IN-1 and incubated at 37° C for 8 h. The cells were lysed, and total RNA was isolated using Tri Reagent (MRC) per the manufacturer’s instructions. First strand cDNA synthesis was carried out using SuperScript II Reverse Transcriptase and random hexanucleotide primers (Invitrogen). The complementary DNA was subsequently used to perform RT-PCR on an iCycler IQ5 Thermal Cycler (BioRad) using SYBR Green (Molecular Probes) with gene-specific primers and JumpStart™ Taq DNA polymerase (Sigma). The crossing threshold value assessed was normalized to β-actin and quantitative changes in mRNA were expressed as fold-change relative to control ± SEM value. The Student’s t-test was used to determine statistical significance. The primer sequences for the genes analyzed are provided in Additional file
1 : Table S1.
7
Real-Time RT-PCR Analysis of Intestinal mRNA
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Total RNA isolated from mouse intestines (24h post-IR) (n = 3 per group and one sample per mouse) was subjected to reverse transcription with Superscript II RNase H—Reverse Transcriptase and random hexanucleotide primers. The complimentary DNA (cDNA) was subsequently used to perform real-time (RT) reverse transcriptase-PCR by SYBR chemistry using gene specific primers and JumpStart Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO). The data was normalized with β -actin. The changes in mRNA were expressed as fold change relative to control with ± SEM value (* p < 0.01) [19 (link)–22 (link)]
The following primers were used:
The following primers were used:
8
Molecular Techniques in Genomic Research
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BNF, SYBR® Green I (10,000× concentration), agarose, JumpStart Taq DNA polymerase, Enhanced Avian RT first-strand synthesis kit (STR-1), GenElute™ PCR Clean-Up Kit, GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit, PCR Low Ladder Marker Set, guanidine thiocyanate, ammonium thiocyanate, Williams’ E medium, LB broth, and LB agar were supplied by Sigma-Aldrich Co (St. Louis, MO, USA). Fluorescein was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Restriction endonucleases were purchased from Fermentas International Inc. (Burlington, Canada). Deoxyribonucleotide triphosphates such as dATP, dGTP, aCTP, and dTTP were provided by Roche Diagnostics (Mannheim, Germany). PCR primers were provided by Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland (oligo.pl), and Genomed, Poland. Agilent RNA 6000 Reagents were provided by Agilent Technologies (Santa Clara, CA, USA). Affymetrix Human Genome U219 Array Strip, GeneChip 3′IVT Express Kit and GeneAtlas Hybridization, and Wash and Stain Kit for 3′IVT Arrays were provided by Affymetrix (Santa Clara, CA, USA). GeneClip™ U1 Hairpin Cloning System—Neomycin Vector and antibiotic G418 (Geneticin)—was provided by Promega (Madison, WI, USA). Lipofectamine 2000 and Opti-MEM® I Reduced Serum Medium was provided by Invitrogen (Carlsbad, CA, USA). All the other compounds were readily available as commercial products.
9
Detect mtDNA m.5789T>C Variant
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The m.5789T>C variant was detected by amplifying a 186-bp PCR product using primers 5694F (5′-AACAGCTAAGCACCCTAATCAACT-3') and 5879R (5′-GAGTGAAGCATTGGACTGTAAATCT-3′). The PCR mix contained 10 ng of DNA, 0.2 U JumpStart Taq DNA polymerase, 0.1 mM of each dNTP, 1× PCR buffer (Sigma-Aldrich, St. Louis), 2.5 mM MgCl2, and 250 nM of each primer in a total volume of 25 µL. Amplification conditions were 95°C for 10 minutes; 35 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 40 seconds; and finally 72°C for 7 minutes. Amplification products were digested with the restriction enzymes TaqαI or Hpy188I (New England Biolabs, Ipswich, MA) using the producer's protocol. TaqαI cuts the wild-type molecules into 2 fragments of 89 bp and 97 bp length, whereas it does not cut the mtDNA molecules carrying the m.5789T>C variant. Hyp188I cuts the wild-type mtDNA into 2 fragments of 58 bp and 128 bp length and the mutated mtDNA into 3 fragments of 58 bp, 31 bp, and 97 bp length. Restriction digestion fragments were separated on a 10% polyacrylamide gel and visualized by SYBR Green I staining (Sigma-Aldrich) and fluorescence detection using a GelDoc system (Bio-Rad Laboratories, Hercules). Proportions of wild-type and mutant mtDNA were estimated from band intensities using ImageJ software. Heteroplasmy values from TaqαI and Hpy188I restriction digestions were averaged.
10
16S rRNA Gene Amplification and Sequencing
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Total DNA was extracted from rectal swabs using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), and a 30-s bead beating step using a Mini-Beadbeater-16 (BioSpec Products, Bartlesville, OK, USA). This genomic DNA was used as the template to amplify the hypervariable V4 region of the 16S rRNA gene using PCR primers (515F/806R with the reverse primers including a 12-bp barcode) and reactions containing: 50 mM Tris (pH 8.3), 500 μg/ml bovine serum albumin (BSA), 2.5 mM MgCl2, 250 μM of each deoxynucleotide triphosphate (dNTP), 400 nM of each primer, 5 μl of DNA template, and 0.25 units of JumpStart Taq DNA polymerase (Sigma-Aldrich, St Louis, MO, USA). Thermal cycling parameters were 94 °C for 5 min; 35 cycles of 94 °C for 20 s, 50 °C for 20 s, and 72 °C for 30 s, followed by 72 °C for 5 min. PCR products were purified using a MinElute 96 UF PCR Purification Kit (Qiagen, Valencia, CA, USA). Libraries were sequenced (1 × 300 bases) using an Illumina MiSeq.
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