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In vitro Transcription kit recommendations?


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#1 Randoramma

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Posted 23 April 2009 - 09:18 AM

So I am currently using Stratagene's RNAmaxx kit followed by Absolutely RNA kit to clean with, but am still getting what appears to be DNA contamination in my standard. This does not appear in my NT controls. Any one have any recommendations for another kit to try that will eliminate the dastardly lingering DNA? I need the very best there is. When you absolutely, positively got to kill every DNA molecule in the room. Accept no substitutes.

#2 mastermi

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Posted 23 April 2009 - 11:42 AM

Have you tried digesting your in vitro transcribed RNA with DNaseI before cleaning?
I don't know any other way to get completely rid of DNA...

#3 Penguin

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Posted 24 April 2009 - 04:28 AM

Ambion make something called TURBO DNAse which is pretty good, also if you treat your sample with Acid Phenol:Chloroform 5:1 (pH 4.5) that should remove any DNA remaining (Ambion's website says "Acidic pH moves DNA into the organic phase").

P

#4 mastermi

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Posted 24 April 2009 - 07:59 AM

also if you treat your sample with Acid Phenol:Chloroform 5:1 (pH 4.5) that should remove any DNA remaining (Ambion's website says "Acidic pH moves DNA into the organic phase").


Acid phenol would be fine for most applications, but usually it's not able to remove every tiny rest of DNA.
If you want your sample to be 100% free of DNA and not only 99,9%, you will have to use DNase.

#5 Randoramma

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Posted 24 April 2009 - 09:22 AM

Have you tried digesting your in vitro transcribed RNA with DNaseI before cleaning?
I don't know any other way to get completely rid of DNA...

Yeah, post transcription I expose my reaction to DNase for 30-60min then proceed to purification.

#6 mastermi

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Posted 24 April 2009 - 10:50 PM

Have you tried digesting your in vitro transcribed RNA with DNaseI before cleaning?
I don't know any other way to get completely rid of DNA...

Yeah, post transcription I expose my reaction to DNase for 30-60min then proceed to purification.


Ok. Then you may test a DNaseI from another supplier. For us, only the one from Fermentas worked fine, whereas for others only the one from NEB worked.
I have no other idea what to do...




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