C1000 touch thermal cycler cfx96 instrument

Manufactured by Bio-Rad

The C1000 Touch thermal cycler CFX96 instrument is a real-time PCR system designed for conducting quantitative PCR (qPCR) experiments. It is capable of precisely controlling and monitoring the temperature of multiple samples during the PCR amplification process.

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Lab products found in correlation

Nucleospin kit, by Macherey-Nagel (3 mentions) Sybr green, by Quantabio (2 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Iq sybr green supermix, by Bio-Rad (1 mentions) Collagen coated 6 well plate, by Corning (1 mentions)

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C1000 Thermal Cycler (1073 citations) C1000 Touch Thermal Cycler (953 citations) CFX96 C1000 Thermal Cycler (58 citations) CFX96 C1000 Touch Thermal Cycler (23 citations) CFX96 Touch Thermal Cycler (22 citations) C1000 Touch Cycler (11 citations) C1000 Thermal Cycler instrument (7 citations) C1000 Touch Thermal Cycler instrument (6 citations) C1000 Touch Bio-Rad CFX96 (2 citations) C1000 Touch Instrument (2 citations)

3 protocols using c1000 touch thermal cycler cfx96 instrument

Quantifying Nascent Transcription in Mouse Hepatocytes

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Total RNA was isolated from primary mouse hepatocytes using NucleoSpin kit (Macherey-Nagel cat# 740955.25) according to the manufacturer's protocol. For qPCR, 1 μg of total RNA was reverse transcribed to generate cDNA (Quantabio cat# 76047–074). qPCR was performed using a C1000 Touch thermal cycler CFX96 instrument (Bio-Rad) using SYBR Green (Quantabio cat# 101414–276). Gene values were normalized with a house keeping gene (Rpl13). The primers indicated ‘nascent’ were designed to amplify nascent transcripts (i.e. the amplified region span exon-intron junctions) as a proxy for transcription and in order to avoid confounding post-transcriptional events. The sequences of primers used in this study are:
Rpl13 - Fwd: AGCCTACCAGAAAGTTTGCTTAC, Rev: GCTTCTTCTTCCGATAGTGCATC
Ahr (nascent) - Fwd: AGGATCGGGGTACCAGTTCA, Rev: ATGTGCCGTATATCAGGCGG
Fh1 (nascent) – Fwd: AGGTGTCGAACTCTACACGGA, Rev: GCTGGTCAGAGTTTGTTTGCTTT
Fosl2 (nascent) – Fwd: CGTCGAATCCGGAGGGAGA, Rev: GCATAATGTCGACCCATGTCC
Gpcpd1 (nascent) – Fwd: CCAGCGCTTCTTCCACTCTC, Rev: GACCCACCTTTGACAAGTCCT
Ppp1r3g (nascent) – Fwd: GGATGCACTTCGCTCGATTG, Rev: ATAGCTTTGATCCACCCCGC
Mt1 (nascent) – Fwd: CTGCTCCACCGGTAAGACTC, Rev: CAAGCCTCTACAACTCGGGG
Nr3c1 – Fwd: CTCCCCCTGGTAGAGACGAA, Rev: TTGACTGTAGCTCCACCCCT
Creb1 – Fwd: TGTAGTTTGACGCGGTGTGT, Rev: TCCACTCTGCTGGTTGTCTG

Goldberg D., Charni-Natan M., Buchshtab N., Bar-Shimon M, & Goldstein I. (2022). Hormone-controlled cooperative binding of transcription factors drives synergistic induction of fasting-regulated genes. Nucleic Acids Research, 50(10), 5528-5544.

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Quantitative PCR of Nascent Transcripts

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Total RNA was isolated from primary mouse hepatocytes using NucleoSpin kit (Macherey-Nagel, Duren, Germany; cat# 740955.25) according to the manufacturer’s protocol. For qPCR, 1 μg total RNA was reverse transcribed to generate cDNA (Quantabio, Beverly, MA; cat# 76047-074). Quantitative PCR was performed using a C1000 Touch thermal cycler CFX96 instrument (Bio-Rad, Hercules, CA) using SYBR Green (Quantabio; 101414-276). Gene values were normalized with a housekeeping gene (Tbp or Rpl13). The primers used in this study (except for Tbp and Rpl13) were designed to amplify nascent transcripts (ie, the amplified region span exon-intron junctions) as a proxy for transcription and to avoid confounding post-transcriptional events. The primers used in this study are detailed in Supplementary Table 4.

Korenfeld N., Finkel M., Buchshtab N., Bar-Shimon M., Charni-Natan M, & Goldstein I. (2021). Fasting Hormones Synergistically Induce Amino Acid Catabolism Genes to Promote Gluconeogenesis. Cellular and Molecular Gastroenterology and Hepatology, 12(3), 1021-1036.

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RNA Isolation and qPCR Workflow

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Total RNA was isolated from collagen-coated 6-well plates (Corning) using NucleoSpin kit (Macherey-Nagel) according to the manufacturer’s protocol.
For qPCR, a 1 µg aliquot of total RNA was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad, 170-8891). qPCR was performed with a C1000 Touch thermal cycler CFX96 instrument (Bio-Rad) using iQ SYBR Green supermix (Bio-Rad, 170-8887). All qPCR experiments were replicated at least three independent times. Error bars represent s.d. of technical replicates. Gene values were normalized with Tbp. The primers used in this study (except for Tbp) were designed to amplify nascent transcripts (i.e., the amplified region span exon-intron junctions) as a proxy for transcription to avoid confounding post-transcriptional events. Due to the highly similar sequence of the Saa1 and Saa2 genes, the primer used in this study could not differentiate between them (and neither did any other primer we tested).

Goldstein I., Paakinaho V., Baek S., Sung M.H, & Hager G.L. (2017). Synergistic gene expression during the acute phase response is characterized by transcription factor assisted loading. Nature Communications, 8, 1849.

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