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.
Tell me more
×
Biology Stack Exchange is a question and answer site for
biology researchers, academics, and students. It's 100% free, no registration required.
|
|
||||
|
|
|
RT-PCR should be able to get you up to ~10kb. If you are finding that it is not working, then you can be 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. |
|||
|
|
