In vitro transcription question - (Mar/26/2012 )

I am trying to make RNA transcripts from a linearized plasmid. I have a question regarding the purification of the template DNA. I have read that you should clean the DNA by phenol/chloroform extraction and other places I have read that DNA from minipreps is fine.

Is it necessary to do a phenol/chloroform extraction prior to in vitro transcription?

I linearized my plasmid with the appropriate restriction enzyme, and then i purified the plasmid using a zymo DNA clean & concentrator spin column.

Will the DNA from the spin column be pure enough for in vitro transcription?

Thanks

I am using Ambion mMessage mMachine kit for in vitro transcription, and I have obtained very good gel results all the time. I directly use linearized plasmids and face no problem. If you obtained good plasmid isolation results and linearized them properly, I don't think you need to take any further steps

kutayozturk on Wed Apr 4 14:20:17 2012 said:

You dont purify the DNA after the restriction digest (to linearize)? I lose a lot of yield when I use the Zymo spin column. I am also getting a smear when I run the reaction on a denaturing gel. The manual suggests treating the DNA with proteinase K. Has anybody had to do this before?

Yes, I don't do any purification after linearization. After checking my linearization results on gel, I directly go on with in-vitro transcription. I have never faced a problem in that way.Can you check your linearization products before and after purification in gel? In this way you can see if the problem is in linearization or in purification. If there's a problem in linearization, you can check with different tubes of enzyme and buffers. There may be some endonuclease contamination from somewhere

I have checked the digest on a gel before purification and it was complete. I have not checked after purification, mainly due to the low yield. The plasmid I am using was from a maxi-prep instead of a mini-prep. Could increased amounts of endonuclease/RNAse be present since it was a maxi-prep? I always did this reaction using mini-prep plasmid in the past.

Well, I always used miniprep, so I am not sure. But there must be an optional step (tells you when working with certain strains having high endonuclease -endA+ for example). If you are skipping it, try to include that step, too. Even if there are some residual endonucleases, it needs time to cause formation of smear on the gels, 1 month kept in -20 for example. Probably that is not the cause of your problem. You may try to check the troubleshooting guide of zymo spin column kit.