I am struggling to get a high enough RNA concentration from transcribed RNA using Invitrogen's MegaScript Sp6 kit. I use Promega's SV total RNA isolation and purification kit to clean up the transcript.
Should I try and elute the RNA in less NFW at the end of purification, even though the kit recommends not doing so? I am currently eluting in 100 microliters.
Or may it be that the transcription reaction is not transcribing DNA into RNA efficiently? I add 1 microliter of 36ng/microliter DNA to my reaction.
If I use more DNA template, will I not struggle to get rid of all the DNA? I use DNaseI enzyme and incubate for 25 minutes. I made a 1:1000 dilution of my DNA template and then only got rid of all the DNA, however I ended up with very little RNA.
What volume would you recommend for elution of RNA?
I don't think removal of DNA is a problem, and for many applications, it may not be necessary to remove DNA, since you have many rounds of transcription in your in vitro transcription reactions, the amount of DNA template in your completed reaction is very small relative to the amount of RNA. I see you use only 36ng of DNA template, which I think is too little.
Here is my answer to the question. To increase the concentration of RNA, Zymo-Spin ™ (Zymo Research) columns whose shape has been revisited do not leave any residual buffer on the surface of the matrices (Figure 1. Zymo-Spin Technology). Thanks to this new column design, it is possible to obtain ultra-pure nucleic acids and at the same time to reduce the elution volume up to 6 μl. These columns not only purify but also concentrate nucleic acids.
Zymo-Spin ™ columns are also excellent for purifying nucleic acids from enzymatic reactions (eg PCR, enzymatic digestion, In-vitro transcription, ChiP, sequencing ...). Nucleic acids are free of all impurities such as enzymes, nucleotides, salts ... and are more concentrated than at the beginning thanks to the reduced elution volume (DNA Clean & Concentrator kits & RNA Clean & Concentrator).
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