OneTaq® RT-PCR Kit combines two powerful mixes, M-MuLV Enzyme Mix and OneTaq Hot Start 2X Master Mix with Standard Buffer for 2-step RT-PCR applications. The two mixes require minimal handling during reaction setup and yet offer consistent and robust RT-PCR reactions.
The first strand cDNA synthesis is achieved by using two optimized mixes, M-MuLV Enzyme Mix and M-MuLV Reaction Mix. M-MuLV Enzyme Mix combines M-MuLV Reverse Transcriptase and murine RNase Inhibitor while M-MuLV Reaction Mix contains dNTPs and an optimized buffer. The kit also contains two optimized primers for reverse transcription and nuclease-free water. An anchored oligo-dT primer [d(T)23VN] forces the primer to anneal to the beginning of the polyA tail. The optimized Random Primer Mix provides random and consistent priming sites covering the entire RNA templates including both mRNAs and non-polyadenylated RNAs.
The amplification step features a OneTaq Hot Start DNA Polymerase in a master mix format. OneTaq Hot Start DNA Polymerase offers higher fidelity than Taq and better amplification. RT-PCR product up to 6 kb can be generated (Figure 1).
Figure 1. First Strand DNA Synthesis
First strand cDNA systhesis was carried out in the presence of 1X M-MuLV Enzyme Mix at 42°C using 0.5 μg of human spleen total RNA in the presence of dT23VN (lanes 1, 4 and 7) or Random Hexamer Mix (lanes 2, 5 and 8). No-RT controls were lanes 3, 6 and 9. OneTaq Hot Start 1X Master Mix was used to amplify a 1.5 kb fragment of beta-actin gene, a 0.6 kb fragment of GAPDH gene, and a 5.5 kb fragment from p532 gene in 35 cycles. The marker lane (M) contains 2-Log DNA Ladder ( NEB #N3200 ).
Figure 2. First Strand DNA Synthesis
The following reagents are supplied with this product:
Store at (°C) Concentration
Oligo d(T) 23 VN -20 50 μM
Nuclease-free Water -20
M-MuLV Enzyme Mix -20 10X
M-MuLV Reaction Mix -20 2X
Random Primer Mix -20 60 μM
One Taq ® Hot Start 2X Master Mix with Standard Buffer -20 2X