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Stratagenes ProSTAR RT-PCR systems use Moloney murine leukemia virus RT (MMLV-RT) for cDNA synthesis of specific target RNAs. The ProSTAR Ultra HF RT-PCR system includes all reagents necessary to carry out cDNA synthesis [MMLV-RT, first-strand buffer, dNTPs, oligo(dT), random primers, and RNase-free water] using total RNA or poly(A)+ RNA. The kit also includes all reagents necessary for subsequent, ultra high-fidelity PCR amplification with PfuTurbo DNA polymerase (PfuTurbo, 10X Ultra-HF PCR buffer, dNTPs). In this system, the cDNA synthesis reagents and protocol provide optimal performance using reaction volumes ranging from 10 to 50 l. Because the kits protocol is versatile, investigators can conserve RNA and cDNA synthesis reagents when a limited number of PCRs are performed (e.g., 10-l volumes) or can scale up the reverse transcription reaction (e.g., 50-l volumes) when multiple targets are amplified from the same cDNA synthesis reaction.
Figure 1
The ProSTAR Ultra HF RT-PCR system has been used to accurately amplify a broad
range of target sizes from a variety of RNA sources, including human,
mouse, yeast, and bacteriophage total RNA or poly(A)+-selected
mRNA. High product yields were achieved for targets ranging in length
from 270 bp to 7.6 kb (Figure
1, Panels A and B). In addition to long target capability, the ProSTAR
Ultra HF RT-PCR system exhibits high sensitivity. RT-PCR products can
be amplified from as little as 1 fg (low comp
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