Overview

Norgen’s 2X One-Step RT-PCR Master Mix is a ready-to-use solution that contains components required for RT-PCR amplification of RNA templates. The mix includes M-MuLV reverse transcriptase, Taq DNA polymerase, dNTPs, reaction buffer, MgCl₂, KCl, and a PCR enhancer/stabilizer. The user needs only to add the template, the primer set and water to the Master Mix to set up the RT-PCR reaction. This convenient 2X One-Step RT-PCR Master Mix reduces the time required to set up PCR reactions and reduces the possibility of contamination, particularly when preparing large numbers of reactions. The optimized master mix allows for robust amplification of RNA templates with high yields of PCR products.

Taq DNA Polymerase is a highly thermostable DNA polymerase that possesses a 5´→ 3´ polymerase activity and a very low 5´→ 3´ exonuclease activity. The source of Taq included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned Taq DNA Polymerase gene from Thermus aquaticus YT-1. M-MuLV Reverse Transcriptase is an RNA-directed DNA polymerase that can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. The source of the Reverse Transcriptase included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned reverse transcriptase gene from M-MuLV.

Norgen’s 2X One-Step RT-PCR Master Mix is available in 3 convenient sizes:

Cat # 28113 - 100 reactions (sufficient for 100 reactions x 20 µL reaction volume)
Cat # 28114 - 200 reactions (sufficient for 200 reactions x 20 µL reaction volume)
Cat # 28115 – 500 reactions (sufficient for 500 reactions x 20 µL reaction volume)

Details

Supporting Data

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Figure 1. Successful Amplification of Different Sized Amplicons. Norgen’s 2X One-Step RT-PCR Master Mix was used in RT-PCR reactions to amplify a number of different amplicons. Lanes 1 and 2 correspond to the E. coli 16s rRNA gene (large amplicon), which is 1.5 kb in size. Lanes 3 and 4 correspond to the E. coli 16s rRNA gene (small amplicon), which is 550 bp in size. Lanes 5 and 6 correspond to the human S15 gene, which is 360 bp in size. Lanes 7 and 8 correspond to the human 5S rRNA gene, which is 120 bp in size. Norgen’s 2X One-Step RT-PCR Master Mix was able to amplify all the different sized products with a high sensitivity and yield.

 

Figure 2. Robust Amplification from Different Amounts of RNA Input. Norgen’s 2X One Step RT-PCR Master Mix was used to amplify the human S15 transcripts from a dilution series of HeLa total RNA isolated using Norgen’s Total RNA Purification Kit. The 361 bp amplicon was robustly amplified from a broad range of RNA inputs, from the ng range down to fg range.

Figure 3. SARS-CoV2 Detection. Performance of Norgen’s 2X One-Step RT-PCR Master Mix (28113) to detect SARS-CoV-2 using Norgen’s 2019-nCoV Primer & Probe Mixes (TM67101) and Norgen’s 2019-nCoV RT-PCR Positive Control (PC67102). The one-step RT-PCR reaction was prepared according to the CDC protocol and the sensitivity of detection of the three targets;  2019-nCoV_ N1 (Circle), 2019-nCoV_ N2 (Triangle) and RNase P (Cross), was compared with Competitor N’s product (Red).

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