Transcript levels of defense-related genes in response to B. cinerea or Pst were analyzed by qPCR. Leaves were ground in liquid N2, and total RNA was extracted with the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, Saint Louis, MO, United States). The isolated RNA was treated with deoxyribonuclease I enzyme (Sigma-Aldrich) to remove remaining DNA. Two micrograms of purified RNA were used for reverse transcription reactions with the Omniscript Reverse Transcription Kit (Qiagen). The qPCR mixture contained 7.5 μL of SYBR Green (Bioline), 5 μL of cDNA (corresponding to 100 ng RNA), and 0.5 μL of 10 μM forward and reverse primers (Supplementary Table 1 ). The final volume was completed with DEPC-treated Water (0.1% diethylpyrocarbonate) to 15 μL. The qPCR was done as follows: 10 min at 95°C initial denaturation and 40 cycles (95°C for 15 s, 60°C for 1 min and 72°C for 30 s). Runs were performed on a MIC qPCR machine (Bio Molecular Systems). Transcript levels were normalized against the expG gene (AT4G26410). The analysis was accomplished based on cycle threshold method (2(–ΔΔCt); Rao et al., 2013 (link)). Three biological replicates were performed for each sample.
Deoxyribonuclease 1 enzyme
Manufactured by Merck Group
Deoxyribonuclease I (DNase I) is an enzyme that catalyzes the hydrolytic cleavage of DNA. It breaks down DNA molecules by hydrolyzing the phosphodiester bonds in the DNA backbone, resulting in the formation of smaller DNA fragments. DNase I is commonly used in various research and laboratory applications that involve DNA manipulation and analysis.
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2 protocols using deoxyribonuclease 1 enzyme
1
Quantifying Defense Gene Expression
2
Transcriptional Response of Defense Genes
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Transcript levels of defense-related genes in response to B. cinerea or Pst were analyzed by qPCR.
Leaves were ground in liquid N 2, and total RNA was extracted with the Spectrum™ Plant Total RNA Kit (Sigma Aldrich, Saint Louis, USA). The isolated RNA was treated with deoxyribonuclease I enzyme (Sigma Aldrich) to remove remaining DNA. Two micrograms of purified RNA were used for reverse transcription reactions with the Omniscript Reverse Transcription Kit (Qiagen, Hilden, Germany). The qPCR mixture contained 7.5 μL of SYBR Green (Bioline, London, UK), 5 μL of cDNA (corresponding to 100 ng RNA), and 0.5 μL of 10 μM forward and reverse primers (Table S1). The final volume was completed with DEPC water to 15 μL. The qPCR was done as follows: 10 min at 95°C initial denaturation and 40 cycles (95°C for 15s, 60°C for 1 min and 72°C for 30s). Runs were performed on a MIC qPCR machine (Bio Molecular Systems, Australia). Transcript levels were normalized against the expG gene (AT4G26410). The analysis was accomplished based on cycle threshold method (2 (-ΔΔCt) ; Rao et al., 2013) . Three biological replicates were performed for each sample.
Leaves were ground in liquid N 2, and total RNA was extracted with the Spectrum™ Plant Total RNA Kit (Sigma Aldrich, Saint Louis, USA). The isolated RNA was treated with deoxyribonuclease I enzyme (Sigma Aldrich) to remove remaining DNA. Two micrograms of purified RNA were used for reverse transcription reactions with the Omniscript Reverse Transcription Kit (Qiagen, Hilden, Germany). The qPCR mixture contained 7.5 μL of SYBR Green (Bioline, London, UK), 5 μL of cDNA (corresponding to 100 ng RNA), and 0.5 μL of 10 μM forward and reverse primers (Table S1). The final volume was completed with DEPC water to 15 μL. The qPCR was done as follows: 10 min at 95°C initial denaturation and 40 cycles (95°C for 15s, 60°C for 1 min and 72°C for 30s). Runs were performed on a MIC qPCR machine (Bio Molecular Systems, Australia). Transcript levels were normalized against the expG gene (AT4G26410). The analysis was accomplished based on cycle threshold method (2 (-ΔΔCt) ; Rao et al., 2013) . Three biological replicates were performed for each sample.
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