Before getting started (general notes)
If transcribing from plasmid:
- This will give you the best RNA with the least number of aborts.
The plasmid must be cut with an enzyme at the end of the desired product so that the transcript is a "run-off" transcript; however, after cutting, it is not necessary to clean the product at all. Just use it in the transcription reaction. If you don't cut, the polymerase just goes around and around making gigantic products you don't want. For plasmid: the least amount of plasmid you can use is about 500ng/40µl transcription reaction. The best amount is about 1000-2000ng/40µl transcription reaction. The rumor is that the sky is the limit on template, that it does not inhibit the reaction when it is very high.
If transcribing from PCR:
- This will give you satisfactory product, but you won't get as much and I often see more aborts.
The PCR product must be cleaned and dNTP's removed before you can transcribe with it. Otherwise, you have a mix of dNTP and rNTP and you can actually make a heterogeneous transcript (not very useful most of the time). You can clean using a Qiagen PCR clean-up kit, or the Qiagen Gel extraction kit (if you have multiple bands), or by doing the phenol/chloroform/ethanol precipitation method. For PCR product: the least amount of PCR product you can use is about 100ng/40µl transcription reaction. The best amount is about 500ng/40µl transcription reaction. The rumor is that the sky is the limit on template, that it does not inhibit the reaction when it is very high.
If transcribing from oligomers:
- This is the least satisfactory of the processes and is limited by the size of an oligo ~100nt.
You will need to design an oligo that has the antisense T7 promoters site as well as an antisense oligo that will make the sense product when transcribed ie:
- 5'- CTGGCCTGCAGCGGGTGGAAGGGGTCGGGGCAGCTGGGGTCTGGGGctcctatagtgagtcgtatta-3'
and a second oligo that binds to the lowercase in the first oligo:
This product will make
with the gag being left over from the promoter site (we have found that ggag helps make more product).
Keep in mind that all transcription reactions results in a great deal of G aborts that could potentially bind to your RNA and inhibit your subsequent reactions. We most of the time use acrylamide gels to purify our RNA. There is also a paper out (Lukavsky and Puglisi, 2004, RNA 10: 889-893) that talks about size exclusion chromatography to make acrylamide free RNA.