( greater accuracy of the Pro...)Performance Comparisons of Commercial RT-PCR Systems

greater accuracy of the ProSTAR ultra HF RT-PCR system is related to the use PfuTurbo DNA polymerase for cDNA amplification. Additional results showed that cDNA produced with different RTs (37-65C) was amplified more accurately with PfuTurbo DNA polymerase than with Tfl, Taq, or Taq-based DNA polymerase blends (Platinum Taq High Fidelity, Advantage KlenTaq Polymerase mix, Expand High Fidelity, LA DNA polymerase blend) (Figure 3 and data not shown). Therefore, PfuTurbo DNA polymerase is the superior choice for carrying out RNA amplification procedures that require the greatest accuracy possible, such as cDNA cloning, sequencing, and/or expression.

Conclusions

Stratagene's ProSTAR RT-PCR systems provide exceptional performance, compared to other commercially available RNA amplification kits. The ProSTAR HF single-tube RT-PCR kit offers the convenience and speed of a one-step format, while consistently producing higher product yields and longer target amplification. The ProSTAR ultra HF RT-PCR system provides superior yield and target-length capability in a versatile two-step format, and the inclusion of PfuTurbo DNA polymerase allows RNA amplification with the greatest accuracy possible.

Methods

RT-PCR reactions were carried out using the recommended protocols for each RNA amplification system/kit. For the ProSTAR HF single-tube RT-PCR system, 50-l RT-PCR reaction mixtures contained 1X HF RT-PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l each), RNA (200 ng of total RNA), 1.25 U of MMLV-RT, and 2.5 U of TaqPlus Precision DNA polymerase. Amplifications were carried out in Stratagenes RoboCycler gradient 96 temperature cycler fitted with a Hot Top assembly, using 200-l thin-walled PCR tubes. The temperature cycling parameters for all tar
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