Total RNA was isolated with the Qiagen RNeasy Mini Kit. Equal amounts of cDNA were synthesized using a High Capacity Reverse Transcription kit (ThermoFisher). TaqMan primers were mixed with TaqMan Universal Master Mix II, with uracil N-glycosylase (ThermoFisher). 18S was amplified as an internal control. QPCR was performed using the Bio-Rad CFX connect system with TaqMan Primer Assays to detect 18S and target genes (ThermoFisher). Primer pair sequences used to detect input and immunoprecipitated DNA sequences for ChIP assay were 5’-CCTGAGAATTGATGGGGAAA and 5’-GTGAACAGGTACCGCACAGA from -329 to -99 of the 5’-flanking RFX6 promoter and 5’-TGACCGGCTTGTCCTTAAAC and 5’-TGAATGGACGGACACTGGTA from -625 to -411 of the 5’-flanking mouse MCT1 promoter.
Cfx connect system
Manufactured by Thermo Fisher Scientific
The CFX Connect system is a real-time PCR detection instrument designed for a variety of nucleic acid amplification applications. It provides precise control of thermal cycling conditions and sensitive fluorescence detection for accurate quantification of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Taqman primer assays, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Uracil n glycosylase, by Thermo Fisher Scientific (1 mentions)
Dynamo sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using cfx connect system
1
Quantitative Gene Expression and ChIP Assay
2
Silencing of Fungal Genes via Protoplast
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These were performed using the protoplast method [67 (link)]. Silencing vectors in initial experiments expressed the open reading frame of PKS1 or GbL1 behind the ham34 promoter in pTEP3, which confers G418 resistance. Experiments with hairpin constructs of PKS1 used pTOR [68 ]. Knocked-down strains were characterized by blot analysis using 32P-labelled probes and RT-qPCR, and confirmed with a minimum of three biological replicates. For RT-qPCR, cDNA was synthesized using the Maxima First-Strand RT-PCR kit (Thermo). Amplifications were performed using the Biorad CFX Connect system using the Dynamo SYBR Green qPCR kit (Thermo) with the following program: 95 °C for 15 min, followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s. Melt curves were generated to evaluate the fidelity of amplification. Expression levels were calculated using the ΔΔCT method, using a constitutive gene (ribosomal protein S3a, PITG_11766) as a control [3 (link), 69 (link)].
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