Complementary DNA (cDNA) was generated from RNA templates. The reaction mix contained 4 µL 5x reaction buffer, 1 µL maxima H minus reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA), 2 µL dNTP nucleotide mix (Roche Diagnostics GmbH, Mannheim, Germany), 0.5 µL RiboLock RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), 5.5 µL nuclease-free water and 5 µL RNA template. PCR reactions were performed using a thermocycler C 1000 (Bio-Rad, Munich, Germany). The PCR temperature profile was: 1) hybridization at 25°C for 10 min, 2) reverse transcription at 50°C for 30 min, 3) enzyme inactivation at 85°C for 5 min, and 4) cooling at 4°C. After cDNA synthesis, the samples were stored at -20°C until further use.
Thermocycler c 1000
Manufactured by Bio-Rad
Sourced in Germany, United States
The Thermocycler C 1000 is a thermal cycler device designed for nucleic acid amplification techniques, such as PCR (Polymerase Chain Reaction). It provides precise temperature control and programmable thermal cycling capabilities to facilitate the various steps involved in DNA or RNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Dntp nucleotide mix, by Roche (1 mentions)
Hotstartaq master mix kit, by Qiagen (1 mentions)
Abi prism 3130, by Thermo Fisher Scientific (1 mentions)
Genemapper v4, by Thermo Fisher Scientific (1 mentions)
Colorless gotaq flexi reaction buffer, by Promega (1 mentions)
Data collection software v3, by Thermo Fisher Scientific (1 mentions)
Universal probe library, by Roche (1 mentions)
4 protocols using thermocycler c 1000
1
Reverse Transcription and cDNA Synthesis
2
OXTR Gene Methylation Analysis
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PCR was performed on bisulfite-converted DNA using touchdown PCRs. For the first target, which covers the first exon of the OXTR gene, a nested PCR was used to amplify the targeted region. For the second target, which is located in the first intron of the OXTR gene, a simple touchdown PCR was performed. For primers and cycling conditions, see Supplementary Table 1 .
All PCRs were performed on a Thermocycler C1000™ (Bio-Rad, Hercules, CA, USA) or a Thermocycler S1000™ (Bio-Rad, Hercules, CA, USA). Amplifying PCRs were performed using the HotStarTaq Master Mix Kit (QIAGEN, Hilden, Germany). Automatic Purification was carried out on a Biomek® NxP using paramagnetic beads (Clean-NGS, GC Biotech®, Waddinxveen, The Netherlands). The concentration of purified DNA was measured by Spectrophotometry using the DeNovix DS-11 Fx + (Denovix, Biozym Scientific GmbH, Hessisch Oldendorf, Germany).
All PCRs were performed on a Thermocycler C1000™ (Bio-Rad, Hercules, CA, USA) or a Thermocycler S1000™ (Bio-Rad, Hercules, CA, USA). Amplifying PCRs were performed using the HotStarTaq Master Mix Kit (QIAGEN, Hilden, Germany). Automatic Purification was carried out on a Biomek® NxP using paramagnetic beads (Clean-NGS, GC Biotech®, Waddinxveen, The Netherlands). The concentration of purified DNA was measured by Spectrophotometry using the DeNovix DS-11 Fx + (Denovix, Biozym Scientific GmbH, Hessisch Oldendorf, Germany).
3
DNA Amplification and Fragment Analysis
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DNA was amplified in the following PCR mixture: Colorless GoTaq Flexi Reaction Buffer (Promega, Madison, WI, USA), 2.5 mM MgCl2, 0.2 mM dNTP, 10% of DMSO, 0.5 U/μL Taq DNA polymerase, 0.5 pmol/μL FAM-labeled Forward primer [(Fam)5′-TGGCGACCCTGGAAAAGCTGAT-3′] and 0.5 pmol/μL Reverse primer (5′-GGTGGCGGCTGTTGCTGCTGCTG-3′). The reactions were run on thermocycler C1000 (Bio-Rad) with the following thermal cycling program: 95 °C for 5 min; then 35 cycles of 95 °C for 15 s, 63 °C for 15 s, and 72 °C for 25 s; with final extension at 72 °C for 25 min. Capillary electrophoresis was performed using capillary genetic analyzer ABI Prism 3130 (Applied Biosystems, Waltham, MA, USA), with size standard Liz 600; the obtained data were analyzed in Data Collection software v3.0 and GeneMapper v.4.0 (Applied Biosystems, Waltham, MA, USA).
4
Fetal and Maternal Placenta Gene Expression Analysis
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Thawed fetal and maternal placenta samples were homogenized (using Precellys®24 tissue homogenizer) and RNA was isolated from using GenElute™ Mammalian Total RNA Miniprep Kit, according to manufacturer’s instructions. RNA yield and purity were measured using a NanoDrop® 1000 Spectrophotometer. The resulting RNA was stored at −80 °C until further processing. cDNA was prepared following the Promega usage information for first-strand cDNA synthesis with M-MLV RT (H-) Point Mutant and the resulting cDNA was stored at −20 °C until further processing. qRT-PCR was performed to measure the relative concentrations of certain genes, using Roche Universal Probe Library and HPRT as house-keeping gene. The LightCycler® Probes Master (Roche) is a ready-to-use reaction mix containing the Taq Polymerase and appropriate buffers. Gene sequences and probes are shown in Supplemental Table 2 . The following qRT-PCR was performed in a Thermocycler C 1000 (Bio-Rad).
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