The OneTaq One-Step RT-PCR Kit offers sensitive and robust end-point detection
of RNA templates. cDNA synthesis and PCR amplification steps are performed in
a single reaction using gene-specific primers, resulting in a streamlined RT-PCR
protocol.
The kit combines three optimized mixes: OneTaq One-Step Enzyme Mix,
OneTaq One-Step Reaction Mix and OneTaq One-Step Quick-Load Reaction
Mix. The Enzyme Mix combines ProtoScript II Reverse Transcriptase, Murine
RNase Inhibitor and OneTaq Hot Start DNA Polymerase. ProtoScript II Reverse
Transcriptase is a mutant M-MuLV reverse transcriptase with reduced RNase
H activity and increased thermostability. OneTaq Hot Start DNA Polymerase is
mixture of a Hot Start Taq DNA Polymerase combined with a proof-reading DNA
polymerase, resulting in high-yield amplification with minimal optimization. The
OneTaq One-Step RT-PCR Kit is capable of amplifying long transcripts up to 9 kb
in length.
Two optimized reaction mixes are included, OneTaq One-Step Reaction Mix and
Quick-Load OneTaq One-Step Reaction Mix. The reaction mixes offer robust
conditions for both cDNA synthesis and PCR amplification. The unique OneTaq
Quick-Load One-Step Reaction Mix contains additional dyes, offering color
indication for reaction setup as well as direct gel loading.
Both total RNA and mRNA can be used as template. The kit can detect a GAPDH
target as low as 0.1 pg per reaction. It can routinely detect RNA targets up to
9 kb. The OneTaq One-Step RT-PCR Kit is capable of multiplex detection of two
or three targets.
Figure 1. Detection of RNA templates of different length.
About 100 ng of Jurkat total RNA was used in 50 μl reactions following the standard protocol. The target sizes were Lane 1: 0.7 kb, Lane 2: 1.1 kb, Lane 3: 1.9 kb, Lane 4: 2.3 kb, Lane 5: 2.5 kb, Lane 6: 5.5 kb, Lane 7: 7.6 kb and Lane 8: 9.3 kb. The marker lane (M) contains Quick-Load 1 kb Plus DNA Ladder (NEB #N0469).Figure 2. Sensitive detection of RNA down to 1 pg.
Serial dilution of HeLa total RNA (Lane 1: 1 μg, Lane 2: 100 ng, Lane 3: 10 ng, Lane 4: 1 ng, Lane 5: 100 pg, Lane 6: 10 pg, Lane 7: 1 pg, Lane 8: 0.1 pg) was used in 50 μl reactions following the Standard Protocol. The marker lane (M) contains Quick-Load 1 kb Plus DNA Ladder (NEB #N0469).
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