Quantitative reverse transcriptase-PCR was performed as previously described.16 (link) RNA was isolated from the cell lines at each passage using TRIZOL (Invitrogen). Complementary DNA was synthesized from 1 mg of total RNA using oligo dT18 primers and Superscript reverse transcriptase (Bioneer, Daejeon, Korea) in a final volume of 20 μl. For standard PCR, 1 μl of first-strand complementary DNA was used as a template for PCR amplification with Taq DNA polymerase (Fermentas, Grand Island, NY, USA). Quantitative reverse transcriptase-PCR reactions were performed using SYBR Green JumpStart Taq ReadyMix (Takara, Shiga, Japan) with an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer). Relative quantification was achieved by normalization to endogenous β-actin. The primers used are shown in Table 1 .
Sybr green jumpstart taq readymix
Manufactured by Takara Bio
Sourced in Japan, United States
SYBR green Jumpstart Taq ReadyMix is a pre-mixed solution that contains the necessary components for real-time PCR, including Taq DNA polymerase, SYBR green, and buffer. The solution is optimized for rapid and efficient DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Superscript reverse transcriptase, by Bioneer (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Exicycler 96 real time quantitative thermal block, by Bioneer (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Icycler iq system, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using sybr green jumpstart taq readymix
1
Quantitative RT-PCR Protocol for Gene Expression Analysis
2
Telomere Length Quantification by qPCR
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Telomeric sequences in immunoprecipitates were evidenced by PCR amplification according to a method described previously [27 (link), 28 (link)]. The final telomere primer concentrations were 270 nM (tel1) and 900 nM (tel2), and PCR amplification was subjected to 35 cycles of 95°C for 15s, 54°C for 2 min. The primer sequences were as following: tel1 5′-GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T-3′and tel2 5′-TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA-3′. Quantitative PCR was done using the SYBR green Jumpstart Taq ReadyMix (TaKaRa) on a Roche LightCycler 480.
3
RNA Extraction and qPCR Analysis
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The total RNA was extracted from the tissues and cells using a Trizol reagent (Ambion. Carlsbad, CA, USA). A total of 1 μg of total RNA was reversely transcripted into cDNA using the GoScriptTM Reverse Transcription system (Promega, Madison, WI, USA) following the manufacturer’s protocol. A quantitative real-time PCR (qPCR) was performed with an iCycler IQ system (Bio-Rad Hercules, CA, USA) using SYBR Green JumpStart Taq Ready Mix (TaKaRa, Shiga, Japan). The relative gene levels were calculated by the 2−ΔΔCT method. Actb mRNA was used as a control. The primers for the qPCR assays are listed in Supplementary Table S2 .
4
Quantitative Analysis of Bcl-2, p53, and NCL
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The final Bcl-2, p53 and NCL primer concentrations were 900nM, and PCR amplification was subjected to 35 cycles of 95°C for 15 sec, 54°C for 2 min. The primer sequences were as following: Bcl-2: (forward) 5-ATGTGTGTGGAGAGCGTCAA-3 , (reverse) 5-TAACTATCCTTGCCCGAACG-3 [9 (link)]; p53: (forward) 5'-AGGTTGGCTCTGACTGTA-3' , (reverse) 5'-CCTCTGTCT-TGAACATGA-3' ; NCL: (forward)5’-GCACTTGGAGTGGTGAATCAA A-3’ , (reverse) 5’-AAATGCATACCCTTTAGATTTGCC-3’ [9 (link)]. Quantitative PCR was done using the SYBR green Jumpstart Taq ReadyMix (TaKaRa) on a Roche LightCycler 480. Reverse transcription involved the Superscript III First Strand Synthesis kit (Invitrogen).
5
Quantitative PCR Protocol for Gene Expression
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PCR amplification was carried out in accordance with the previously described method. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. cDNA was then reverse transcribed according to the manufacturer’s instructions. Quantitative PCR was carried out using a SYBR green Jumpstart Taq ReadyMix (TaKaRa) on a Roche LightCycler 480. A threshold cycle for each PCR amplification was subjected to 35 cycles of 95°C for 15 s and 54°C for 2 min. The primer sequences and their conditions for use were summarized in Table 2 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize expression levels.
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