( leased. This novel technology increases...)cMaster RTplusPCR System for increased sensitivity and product length ,,, in RT-PCR

leased. This novel technology increases the sensitivity and specificity of cMaster RTplusPCR. A single kit combines One- and Two Step RT-PCR protocols with high dynamic detection ranges and broad ranges of PCR product size (up to 5.3 kb for One Step reactions; up to 12.3 kb for Two Step protocols). The following experiments were designed to demonstrate the use of the kit in various applications.

The first experiment focuses on the extraordinary sensitivity of the new system with respect to One Step RT-PCR. Working with small amounts of target material is the most critical factor in One Step applications. Performing long range RT-PCR in a One Step protocol is no longer a problem as demonstrated in experiment two. The novel buffer system in the kit allows optimal processivity of both the reverse transcriptase and proofreading polymerase mix. The optional Two Step RT-PCR protocol provided in the system is adaptable to a wide range of templates. Experiment three depicts amplification of various templates. Whether moderately sized or very long, low- or high abundance, the new system offers a comprehensive
solution for all RT targets.

Materials and Methods

Eppendorf cMaster RTplusPCR System
Eppendorf Perfect RNA Eukaryotic Kit
All PCR* reactions were performed on the Eppendorf Mastercycler gradient.

Experiment 1: One Step, Sensitivity Test

Target for RT-PCR amplification: human Alpha Tubulin mRNA, a 500 fragment. Template RNA: total RNA purified from human HELA cells. Template concentrations in the reactions ranged from 1 g down to 10 pg. Protocol: Eppendorf cMaster RTplusPCR System, standard One Step RT-PCR protocol. 50 l react
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