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DNase treatment for RNA purification - (Apr/23/2004 )

Hi

I isolated total bacterial RNA and treated with DNase once time, and then suspended it into TE buffer, but the gel-running showed that there are still some contaminating DNA in it, so it is necessary to treat it with DNase. It seems that DNase reaction solution dont contain TE buffer, just RNase-free water, who would like tell me how to do it ?

-weimin42-

Hello Weimin42,

I don't understand why you didn't get rid of DNA after DNase I treatment, have you followed the right procedures?

QUOTE
It seems that DNase reaction solution dont contain TE buffer, just RNase-free water, who would like tell me how to do it
.
Not sure what you are saying rolleyes.gif

Check here at Ambion http://www.ambion.com/techlib/tb/tb_181.html.
Three methods of removing DNA contamination are described as follows.
DNase digestion
DNase is an endonuclease that cleaves DNA by breaking phosphodiester bonds. It must be inactivated or removed from the reaction prior to PCR, otherwise, it may digest newly amplified DNA. For this study, we tested 2 concentrations of DNase I (10 and 50 ┬Ál DNase I/ml sample) and four DNase I inactivation/removal methods:

Chelation with 20 mM EDTA
Heating at 70C for 5 minutes
Proteinase K digestion followed by phenol/chloroform extraction and NH4OAc/EtOH precipitation
Protein removal using Ambion's RNAqueous Kit
Acid phenol:chloroform extraction
Acid phenol:chloroform (5:1 phenol:CHCl3; pH 4.7) extraction partitions DNA into the organic phase. The RNA remains in the aqueous phase and can be subsequently recovered by precipitation.

Lithium chloride (LiCl) precipitation
LiCl precipitation is a selective precipitant of RNA. It inefficiently precipitates DNA which is discarded in the supernatant.

-labrat-

Hi ,Veteran

Thanks for your reply.

Certainly, i did get rid of DNA after DNase I treatment, but i was not sure whether the DNA contaminations still extist. The results of gel-running proved

there were still some DNA in my isolated RNA, so i want to treat it with DNase I once more, what i mean is that whether i can use TE buffer(pH 8.0) in the

DNase I reaction instead of RNase-free water because i suspended the fist-DNase I- treated RNA in the TE buffer.

Thank you for your reading.

weimin

-weimin42-

I would suggest that the on column DNAseI digest kits are actually not as efficient as manufacturers say, and that can mean that you have to treat your RNA 2/3 times.
If you are doing RT-PCR you can carry out the DNAseI digest in a 20ul reaction using your PCR / RT-PCR buffer. Do this in PCR tubes in a thermal cycler. It is sensible (but not essential) to add EDTA to ~1.5 mM (final) because you will need to heat inactivate your DNaseI at 75 C for 10 min. The EDTA helps to protect your RNA at high temperatures. EDTA will take some of the Mg out of the action, but when you add the rest of your reaction mix (below) it will get diluted down and you will add extra Mg in your PCR buffer anyway.
Then simply make a mastermix of the rest of your RT-PCR components such that you can add 30 ul to your DNAseI treated samples to get a 50ul RT-PCR mix.
Then run your RT-PCR.
It is important to always carry out 'no RT' controls with cheapish Taq to check the DNA content of your RNA.

-tuckern-

Dear weimin...

Are you doing RNA extraction? From my understanding of what you have posted, you are. Based on my understanding, we need to be very careful when extracting RNA as RNA degrades easily. Tris cannot be autoclaved, therefore there is possibility of RNase contamination in TE buffer (pls correct me if I'm wrong...). So, normally during RNA extraction, we should be using RNase-free water, as in DEPC-treated dH2O or something like that. As for the purity of your RNA sample, maybe you can try UV spectrometry as well. Try reading the protocol that comes with your DNase I pack. I'm sure you should be able to gain some insight regarding how long you should incubate your sample with DNase I and also how to eliminate the DNase I after the reaction. Always remember, the downstream application of your RNA will determine how to isolate your RNA. Anyway, keep in mind that your RNA can degrade easily...

Cheers! cool.gif

-kherxing-

Tris can certainly be autoclaved. But it cannot be treated with DEPC, which is perhaps what you meant to say. RNAse contamination of the buffers certainly is an important consideration.

-phage434-

For an DNase enzyme to work efficiently it needs both magnesium and calcium ions and the correct pH

I woudl recommend the following buffer (final conc)
40mM Tris pH8.0
10mM MgCl2
3mM CaCl2

With incubation at RT or 37C

DNase will not work in water alone

-John Buckels-