I'm currently using the RiboMax T7 transcription kit from Promega and I am able to get transcripts from my DNA template of 1.6 kb but am having difficulty getting the longer transcripts. I am analyzing my transcripts via bioanalyzer and it appears as though the 3.6 kb and 6.5 kb reactions are giving me various products that are much shorter (starting with 25 bp, then 200 bp, 1000 bp, and finally a 4 kb for the 6.5 kb transcript, the peaks are pretty uniform between samples). Is there a kit that is better for longer transcripts or are there reaction conditions I can vary to get these full length transcripts? Thank you in advance.
Have you tried supplementing with GTP? Usually GTP becomes limiting with in vitro transcription. The mMessage machine kit from Ambion (now Life Technologies) has tips for optimizing transcription of large (>5 kb) transcripts. It might be worth checking it out as I have had good results with this in the past.
J
Abortive transcripts (short transcripts happening prior to promoter escape during transcription initiation) will be produced with any RNA polymerase (T7, bacterial, eukaryotic); you will not be able to prevent their production. Their pattern of the transcripts are usually consistent. I don't know about the 4kb one, except to guess the possibility of an ulternate promoter-like region within the template.
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