QIAquick PCR purification kit for in vitro transcription? - (Aug/12/2008 )
Im wondering if somebody has any experience in using the QIAquick PCR purification (Quiagen) kit for cleaning up DNA in purpose of performing an in vitro transcription.
Usually, Im using the standart protocol performing a phenol and chloroform extraction with a final EtOH precipitation to clean up my linearized plasmids. Now we got that kit in the lab, and it seems to be possible to save a lot of time using the kit. It would be great to avoid the time consuming phenol/chloroform method. But I dont know if the quality of the purified DNA is good enough to perform an in vitro transcription.
According to the manufacturer, it should be possible to purifiy enzyme-treated DNA for further use as in vitro transcription or even microinjection. Does anybody has experience in using this kit for this purpose, and what are the advantages compared to the classical phenol/chloroform method?
I had to make the same decision about one week ago. i have exactly the same kit here.
i think it denpeds on your source of DNA. i think it will work from PCR reactions and i would definitely give it a try.
the problem with plasmid DNA is that it gets a heavy RNase treatment during isolation and i don't think that you can remove them completely with that kit especially if they are sticking to your DNA.
I decided for the safe way of proteinase K /SDS treatment followed by PCl extraction and EtOH precipitation.
But if you try it please share your result
thanks for your quick answer. I have to say that after my Midi preps, I always extract and precipitate the plasmids using Roti-Phenol, Chloroform and EtOH to clean them up. I think after a Midi prep, the plasmids are not "clean" at all. The linearization works then better too.
But Im gonna try the cleanup of the linearized plasmids with the kit today, and tomorrow Ill do the in vitro transcription. I will post the results As I said, Quiagen writes in the handbook that this kit can be used to clean enzyme-treated DNA, resulting in a yield which can be used for in vitro transcription.... lets hope the best
ok, the in vitro transcription worked but the quality is not that good... I did not include a sample of the "classical" cleaned DNA, so I cannot compare, but the quantification showed around 10 ug/ul, which is not optimal.
I dont know if the RNA is convenient for my virus inoculations... this will come out later.
ok, i will stick to the "old school" protocol. moreover, i don't have to do so many samples. but isn't 10µg/µl a quite huge amount? i'm doing IVT with ambion's mMessage mMachine kit for oocyte microinjection experiments and i got ~500-600ng/µl of yield. and that is already the double amount which ambion predicts. I have purified the RNA with the Megaclear Kit, eluted in 100µl (preheated) elution solution and measured on two different photometers, eppendorf biophotometer and nanodrop.
thanks for the information!
i only used a photometer for measurement, and I think the remaining nucleotides disturbed this measurement... on a gel, I get bands, but they are quite fainting.
I need as much RNA as possible, because I use it for virus inoculations After transcription, I dont process the transcripts further because they are infectous anyway. But I think the next time I will choose another kit from another manufacturer to try out. I would definitly not take the Quiagen kit.
you can try to just add this proteinase K treatment step before the qiagen purification, so any residual RNase which you would carry over in your IVT should be inactivated. or you just end up with proteinase K in your IVT instead of RNase, which i guess is also not optimal