I want to sequence a 12kb mRNA. I extracted total RNA by using the RnxPlus kit and cDNA syntheses was carried out using the qiagen kit. I don't have any experience sequencing an mRNA with a 12kb length. I need to find a way to sequence only one mRNA. Any thoughts would be welcome.
If you want only mRNA in your sample, you can either use poly-T columns to enrich them in sample or use anchored poly-T primers for cDNA generation.
you can try reverse transcription, pcr for large scale cDNA with specific primers, and then use some specific sequence primers, one primer is okey for almost 700bp or so, you need many sequence primers for different parts
I don't have the experience, but have suggestions as follows that may work: Use long-rang reverse transcriptase and polymerase to generate cDNA, run sample and marker DNA in agarose gel, cut the gel at around 12 kb, recover the sample, and sequence it.
- Badges
- Science method