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Has anyone optimized RT for long transcripts (9kb)? The downstream application will be PCR amplification and Illumina library prep. It will be trivial to make internal primers sets for the PCR that are specific as long as there are no chimeric sequences. If there are, they will probably get primed also. If anyone knows of an optimization and/or other potential pitfalls, I would love to hear them.

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RT-PCR should be able to get you up to ~10kb. If you are finding that it is not working, then you can by long-range RT-PCR kits commercially (look at Stratagene and Qiagen, for example). The Qiagen can handle up to 12.5 kb in a two-step RT-PCR system.

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With longer sequences, the dNTPs become limiting. Try to add more dNTPs while keeping the amount of primer the same. Don't be afraid to run several reactions at different amounts of reagent to find the optimal amount. Also, increase your reaction time. The reverse transcriptase and DNA polymerase need more time to cover a longer transcript.

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