I have an aptamer within a DNA plasmid that I use for fluorescence experiments. When transcribed into RNA, the aptamer has a specific fluorophore it interacts with. I'm wanting to get Kd data of the aptamer and it's fluorophore, but need to transcribe, isolate and purify the RNA from the DNA plasmid I have in stock.
Any recommendations on the best way to go about this?
You can use any common in vitro transcription kit to do this, if I understand your aims correctly. If you are feeling ambitious, you can skip the kit and go a homebrew route as many users do. Good luck!
How big is the RNA molecule you're after? It might be faster and cheaper to buy it as an RNA oligo rather than go down the in vitro transcription route.
Philip Kitchen The aptamer itself is ~98 nucleotides? I've ordered a plasmid that contains simply that aptamer and T7 promoter so I can isolate simply the RNA.
Edit: Thanks for your reply + help!
Craig Silver I see, and would I carry out a purification and isolation protocol after the transcription, as the aptamer is contained within a bacterial plasmid?
For purification and isolation after the transcription, you just run your sample through a small mini elute spin column. You can add DNAse to remove plasmid, EDTA to inactivate the DNAse, wash wash wash, and elute in your buffer of choice. A full protocol would be available in the kit. A more difficult but still doable method would be to do an ethanol precipitation and isolate a pellet of your RNA, but that is better left for advanced users, the spin columns are fool proof. Since your RNA is already behind a T7 promoter, you can just order any T7 in vitro transcription kit, and you'd have your RNA in a day.
If you went this route, you would be able to make more than you would ever need, versus ordering RNA oligos.
Craig is right - in vitro transcription kits give more or less the protocol to follow when performing this step, so whether you buy them or not, you can just check on line for their protocols. He's also right that you could add DNAse and clean up with a miniprep column since the RNA oligo is quite big and will stick to the columns while the chewed up DNA will pass through. If you want to get old school - PCR the fragment starting with T7 site and ending with DNA base that is last for the RNA you want to transcribe and use this directly for in vitro transcription. Isopropanol/Ethanol precipitate the whole reaction to quench, then add buffer and nuclease. Then ethanol precipitate again, add salt, then phenol/chloroform extract your RNA and finally...one more isoprop or ethanol precipitation to remove the chloroform and any remnant small DNA fragments.
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