( tectable in 100 and 10 ng of t...)cMaster RTplusPCR System for increased sensitivity and product length ,,, in RT-PCR

tectable in 100 and 10 ng of total RNA using the One Step RT-PCR protocol. This is a very difficult PCR at best, and most manufacturers of RT-PCR kits do not even publish results of one-step protocols on templates this long. In contrast the Eppendorf kit can consistently and reliably amplify this product from 10 ng of RNA from HELA cells.

Experiment 3: Two Step RT-PCR of short, long, rare and abundant transcripts from various sources of total RNA.



Fig. 3: Two Step RT-PCR of multiple targets and RNA sources. M: DNA Ladder Mix 0.110 kb (MBI Fermentas).

RT-PCR was performed on five genes that exhibit various levels of expression. In order to maximize sensitivity the RT reaction was performed separately from the PCR reaction. In the case of Alpha Tubulin and TNFR1, we were able to use a two-step protocol and combined the annealing and elongation step into a single 68 C step. As shown in fig. 3, all reactions worked well regardless of fragment length or copy number. Even Dynein, an extremely rare transcript that is
very long (12.3 kb), was efficiently amplified with the cMaster RTplusPCR. These results were extremely reproducible and show that the Eppendorf kit can be expected to give reliable results for difficult RT-PCR reactions.

Details for Fig. 3



Discussion

The cMaster RT is designed to provide maximum flexibility for all demanding RT and RT-PCR applications. A recombinant homo-dimeric viral reverse transcriptase together with a unique formulation of the RTplusPCR buffer allows efficient cDNA synthesis over a broad temperature range (37 C60 C)
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