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Megaplex RT Primer Pools: cDNA Synth for Real-Time PCR

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#1 eso_meso



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Posted 19 October 2009 - 09:40 AM

I have used these for large-scale discovery but am attempting to prepare cDNA stock with the primer pools for use with single taqman primer PCR. Anyone have suggestions on procedure? For those who don't know, Megaplex primer pools are a 10X mixture of hundreds of ABI's miRNA assays RT primers for use with a 384-well array card with embedded taqman PCR primer-probes. The Megaplex protocol calls for 350-1000 ng starting total RNA while the miRNA Assays protocol calls for 1-10 ng. I am assuming the difference is due to the requirement for more transcript to bind individual primers. The cycling conditions are as follows: (40 cycles) 1) 16C/2 min, 2) 42C/1 min, 3) 50C/1 sec, Hold 85C for 5 min. I plan to use the miRNA Assays PCR protocol with 100 ng template.

Additionally, the total volume/well for Megaplex RT is 7.5 uL with 1.5 uL RTase. I plan to up it to 22.5 (x3) using 3000 ng template and really don't want it to degrade. Is an 85C for 5 min step sufficient to inactivate this 4.5 uL of RTase?


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