Cfx connect real time machine

Manufactured by Bio-Rad

The CFX Connect Real-Time PCR Detection System is a thermal cycler that enables real-time quantitative PCR (qPCR) analysis. It provides precise temperature control and detection capabilities for sensitive and reliable gene expression analysis, genotyping, and other real-time PCR applications.

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Lab products found in correlation

Superscript 3, by Thermo Fisher Scientific (2 mentions) Sybr green master mix, by Bio-Rad (2 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Sybr green, by Bio-Rad (1 mentions) Iscript kit, by Bio-Rad (1 mentions) Nanodrop spectrometer, by Thermo Fisher Scientific (1 mentions) Taqman primer, by Thermo Fisher Scientific (1 mentions) Lysing matrix a tube, by MP Biomedicals (1 mentions) Rnaprotect tissue reagent, by Qiagen (1 mentions) Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions) Maxima first strand cdna synthesis kit for rt qpcr, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

CFX Connect (733 citations) CFX Connect Real-Time PCR machine (60 citations) CFX Connect machine (42 citations) CFX machine (16 citations) CFX Connect Real-Time System machine (13 citations) CFX Connect™ Real-Time qPCR machine (9 citations) CFX Connect Real-Time PCR detection machine (4 citations) CFX Connect Real-Time PCR Detection System machine (2 citations)

6 protocols using cfx connect real time machine

DNA Extraction and qPCR Analysis

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Tissues were harvested from animals and immediately frozen in liquid nitrogen. Tissues were cold processed with pestle and mortar and then genomic DNA was isolated using Quiagen’s DNeasy Blood and Tissue kit. DNA concentration was measured in quadruplicate and then serially diluted to 3 picograms/μl. Two μl of diluted genomic DNA was then loaded into SYBR Green Master Mix reaction and assayed using mL1 primers on BioRad CFX Connect Real Time machine.

Simon M., Meter M.V., Ablaeva J., Ke Z., Gonzalez R.S., Taguchi T., Cecco M.D., Leonova K.I., Kogan V., Helfand S.L., Neretti N., Roichman A., Cohen H.Y., Meer M.V., Gladyshev V.N., Antoch M.P., Gudkov A.V., Sedivy J.M., Seluanov A, & Gorbunova V. (2019). LINE1 derepression in aged wild type and SIRT6 deficient mice drives inflammation. Cell metabolism, 29(4), 871-885.e5.

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SIRT6 Thermal Shift Assay

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Four μM SIRT6 protein was combined with 1x SYPRO Orange dye in storage buffer (50 mM Tris–HCl pH = 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol) to 50 μl. Samples were placed in a qRT–PCR plate and run on a BioRad CFX Connect Real‐Time machine using a Melt Curve protocol (30°C‐75°C, 0.5°C steps in 10 s intervals).

Simon M., Yang J., Gigas J., Earley E.J., Hillpot E., Zhang L., Zagorulya M., Tombline G., Gilbert M., Yuen S.L., Pope A., Van Meter M., Emmrich S., Firsanov D., Athreya A., Biashad S.A., Han J., Ryu S., Tare A., Zhu Y., Hudgins A., Atzmon G., Barzilai N., Wolfe A., Moody K., Garcia B.A., Thomas D.D., Robbins P.D., Vijg J., Seluanov A., Suh Y, & Gorbunova V. (2022). A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A. The EMBO Journal, 41(21), e110393.

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Quantifying Osteogenic Gene Expression

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When the cells were fused to 80%, Trizol Reagent was used to isolate the total RNA, and then the cells were treated with DNase. cDNA was synthesized using Superscript III (Life Technologies) cDNA kit with the Random Hexamer primer. SYBR Green Master Mix (BioRad) was used to perform PCR on the BioRad CFX Connect Real Time machine using 30 ng of cDNA per reaction with 4x reactions. Denaturation, annealing, and extension were performed under the following conditions: 95^°C for 30 sec, 95^°C for 5 sec for a total of 45 cycles, and 60^°C for 20 sec. Relative gene expression was analyzed by the 2^−ΔΔCt method [18 (link)] and standardized by the GAPDH level. The primers were listed as below:
SIRT6 F: 5’-GCAGTCTTCCAGTGTGGTGT- 3’, SIRT6 R: 5’-GATCTGCAGCGATGTACCCA- 3’.
Osteocalcin (OCN) F: 5’-GCAGTCTTCCAGTGTGGTGT-3’, OCN R: 5’-GATCTGCAGCGATGTACCCA- 3’.
RUNX2 F: 5’- CGCCTCACAAACAACCACAG- 3’, RUNX2 R: 5’- TCACTGTGCTGAAGAGGCTG- 3’.
BMP2 F: 5’- ACTCGAAATTCCCCGTGACC- 3’, BMP2 R: 5’- CCACTTCCACCACGAATCCA- 3’.
KLF5 F: 5’- ACGCTTGGCCTATAACTTGGTT- 3’, KLF5 R: 5’- TGATGTGTGTTACGCACGGT- 3’.
GAPDH F: 5’-AATGGGCAGCCGTTAGGAAA- 3’, GAPDH R: 5’-GCGCCCAATACGACCAAATC- 3’.

Li C., Xiao F., Wen Y., Wu J, & Huang N. (2022). Krüppel-like factor 5 -mediated Sirtuin6 promotes osteogenic differentiation and inhibits inflammatory injury of lipopolysaccharide-induced periodontal membrane stem cells by inhibiting nuclear factor kappa-B pathway. Bioengineered, 13(3), 6966-6977.

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Quantifying Full-Length L1 Transcripts

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Total RNA was isolated from cells at 80% confluence using Trizol Reagent and then treated with DNase. cDNA was synthesized using Superscript III (Life Technologies) cDNA kit with the Random Hexamer primer. qRT-PCR was performed on the BioRad CFX Connect Real Time machine with SYBR Green Master Mix (BioRad) using 30 ng of cDNA per reaction with 4x reactions/sample. Probes targeting a conserved region of the L1MdA1 family the first 108 bp of ORF1 coding region were used to assess full length L1 transcripts. Additonal primers targeting bp 2288–2430 of the L1MdA ORF2 reading frame were assessed, along with primers to the L1MdTf family ORF1 and ORF2 reading frames (bp 706–900 and bp 3362–3536, respectively). All primer sets were tested for specificity and efficiency. All primer sets produced comparable results and expression trends equivalent to the L1MdA ORF1 primer pair. Efficiency verified L1MdA1 (mL1) primers previously described were used to assess L1 transcript abundance standardized to QuantumRNA Actin Universal primers (Van Meter et al., 2014 (link)). All primers can be found in Key Resources Table.

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Zebrafish Heart Gene Expression Analysis

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For total RNA extraction, 400-600 36 hpf Tg(myl7:GFP) transgenic embryos were euthanized using tricaine. Hearts were dissociated in L-15 media using gel loading pipette tips, and GFP fluorescence was used to manually isolate the population (Lombardo et al., 2015 (link)). The isolated hearts were briefly washed with L-15 media supplemented with 10% BSA and total RNA was extracted using Trizol (Ambion) with phenol/chloroform extraction. cDNA was synthesized using the iScript Kit (BIO-RAD, catalogue number: 1708841) using 1,000 ng of RNA per 20 μL reaction. qRT-PCR was performed using SYBR green (Bio-Rad) using CFX Connect Real-Time machine with a minimum of three independent biological and technical samples. The following primers were used:
ccnd1 F: 5’-TGGGATCTGGCCTCAGTGAC-3’
ccnd1 R: 5’-TGAAGTTGACGTCTGTCGCAC-3’
ccnd2a F: 5’-AGCCGTATTAAAGGTCGAAAAGG-3’
ccnd2a R: 5’-CCTCGCAGACCTCTAACATCCA-3’
ccnd2b F: 5’-ACTGCTGTGGGAGTTGGTGG-3’
ccnd2b R: 5’-AAGGTTTGCGTGTGCTTGCG-3’
ccnd3 F: 5’-CATCGCCCTCACGGCTACAG-3’
ccnd3 R: 5’-ACATGCAGAGAACGCCTTGTCC-3’
ccne1 F: 5’-TCAGGGCTGAAGTGGTGTGA-3’
ccne1 R: 5’-GGAGTGAACCTTTCCCAGCC-3’
ccne2 F: 5’-GCACTGGACACTGCGGACAA-3’
ccne2 R: 5’-GGGACTCTTCTATTGCACTCGCC-3’
nkx2.5 F: 5’-CCGGATCCTCTCTTCAGCG-3’
nkx2.5 R: 5’-CCTGACAAAACCCGATGTCTTT-3’
nkx2.7 F: 5’-GCTTCAGTGTATGCAGAACACCC-3’
nkx2.7 R: 5’-CGGGGCCGAAAGGTATCTCTGC-3’

Sarti A., Malladi R, & Sethian J.A. (2000). Subjective surfaces: A method for completing missing boundaries. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6258-6263.

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Gene Expression Analysis of Immune Cells

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Tissues were stored in RNAprotect Tissue Reagent (Qiagen, 76106) at -80°C until processing. They were lysed with a FastPrep 5G (MP Biomedicals) in lysing matrix A tubes (MP Biomedicals, 6910050). mRNA was extracted with the RNeasy minikit (Qiagen, 74106), according to the manufacturer’s protocol, and quantified with Nanodrop spectrometer (Thermo Fisher Scientific). cDNA was synthesized from mRNA with the Maxima First-Strand cDNA synthesis kit for RT-qPCR (Thermo Fisher Scientific, K1642) according to the manufacturer’s protocol. qPCR reactions were run on a CFX Connect Real-Time machine (Bio-Rad) with Taqman universal master mix II (Thermo Fisher Scientific, 4440040) and Taqman primers (Thermo Fisher Scientific: Gapdh, Mm99999915_g1; Ncr1, Mm01337324_g1; Ifn-γ, Mm01168134_m1; Gzmb, Mm00442834_m1). Relative gene expression was quantified and expressed as 2^-ΔCt, with Gapdh as an endogenous control.

Demaria O., Gauthier L., Vetizou M., Blanchard Alvarez A., Vagne C., Habif G., Batista L., Baron W., Belaïd N., Girard-Madoux M., Cesari C., Caratini M., Bosco F., Benac O., Lopez J., Fenis A., Galluso J., Trichard S., Carrette B., Carrette F., Maguer A., Jaubert S., Sansaloni A., Letay-Drouet R., Kosthowa C., Lovera N., Dujardin A., Chanuc F., Le Van M., Bokobza S., Jarmuzynski N., Fos C., Gourdin N., Remark R., Lechevallier E., Fakhry N., Salas S., Deville J.L., Le Grand R., Bonnafous C., Vollmy L., Represa A., Carpentier S., Rossi B., Morel A., Cornen S., Perrot I., Morel Y, & Vivier E. (2022). Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant. Cell Reports Medicine, 3(10), 100783.

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