Hi everybody!
I'm looking for a good protocol to extract DNA from Trizol samples.
I usually extraxt RNA with Trizol, but now I'd like to take also gDNA.
I used this protocol:
1. After having taken the aqueous phase with RNA, spin down the tubes which contain the interphase/organic phase with TRIzol at 12,000 x g for 5 min. at 4 C. Carefully remove any remaining aqueous phase which would contaminate your DNA sample with RNA.
2. Add 0.25-5 ml of the BEB (back extraction buffer: Guanidine Thiocyanate 4 M, Sodium Citrate 50 mM,Tris (free base) 1M) per 1 ml of TRIzol used for the RNA extraction to each tube. Mix intensively at least 3 min. by inversion or put on a shaker for 10 min.
3. Spin tubes at 12,000 x g for 30 minutes at room temperature (I tried also at 4°C).
4. Transfer upper aqueous phase and eventually save interphase for protein isolation.
5. Add 0.4 ml isopropanol per 1 ml TRIzol used for original RNA isolation. Mix and incubate for 5 min. at RT.
6. Spin samples at 12,000 x g for 15 min. at 4 C.
7. Remove supernatant, keep pellet with DNA. Add 0.5 ml of ethanol 70% per 1 ml TRIzol used for RNA extraction and wash pellet by inversion.
8. Spin down at 12,000 g for 15 min. at 4 C. Remove ethanol and dissolve pellet in about 400 ul of 1x TE buffer. DNA can be stored at 4 C.
But...The acqueos phase is cloudy and pink. I can't see the pellet (if a DNA pellet is expected) and after resuspension I've nothing in the eppendorf tube.
Any idea/suggestions?
tnx in advance
Trizol is fine for total RNA, but I've never been very succesful with DNA or proteins using their protocol. If you want really clean gDNA you should use a more classical protocol.
Hello Nicola,
What are you trying to extract DNA? Plants, bacteria ...? It has plants that have high levels of contaminants (polyphenols and polysaccharides) that prevent recovering nucleic acid quality and quantity through the TRIzol method. Maybe it's recommended you use a specific protocol for the DNA sample, for example: of Doyle & Doyle or Dellaporta.
Using TRIzol reagent, the aqueous phase is really pink. The bad is it getting dark, it would be a sign of rust and you lose all the quality, quantity and integrity of nucleic acids.
His intention in addition to extracting RNA of perfect quality, it is also extracting DNA at the same time on the same sample? It has several specific protocols to isolate DNA or RNA. Depending on your goal, the TRIzol is not appropriate.
Links:
http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb21/r03-032.pdf
Dellaporta SL, Wood J, and Hicks JB (1983) A plant DNA minipreparation: version II.
Plant Mol Biol Rep 1: 19-21.
Doyle JJ and Doyle JL (1987) A rapid DNA isolation procedure for small quantities of
fresh leaf tissue. Phytochem Bull 19: 11-15.
Good luck,
Marcos
Fantastic! so...I've definitively to change method...
The aim of my experiment was to analyze both RNA and DNA...from the same sample...
And...my samples are human cells...
Nicola,
TRIzol reagent or TRI Reagent is recommended for possibly that you are trying (http://www.mrcgene.com/tri.htm).
You should be careful in the proportions of TRIzol per amount of sample. TRIzol purification will cause little inappropriate. Always keep your samples on ice before and during and following the protocol carefully. Use sterile equipment, as tips and tubes or free of RNase and prepare new solutions with high quality reagents. The precipitation stage, keep the tubes in a temperature around 0 degrees, it will help to precipitate the DNA and/or RNA, but without exaggeration of time and temperature, as contaminants may also precipitate. Use the correct spin speed and times. The pellet of DNA or RNA is not always visible, especially RNA. And darken, oxidation means, hence I suggest replacing the method of isolation.
In this articicle:
Simultaneous Isolation of DNA and RNA from the Same Cell Population Obtained by Laser Capture Microdissection for Genome and Transcriptome Profiling. Journal of Molecular Diagnostics 2008, Vol. 10, No. 2
http://jmd.amjpathol.org/cgi/content/full/10/2/129
Little can help you, because I work with Molecular Biology of Plants.
I wish good luck,
Marcos
tnx Marcos, for RNA is exactly what I do...(and I always see RNA pellet...)
The link you provided me is the "on-line version" of Tri-ragent/Trizol (they are the same) datasheet...
I can try with the "classical" precipitation: phenol phase and interphase + 0.3 ml ethanol and DNA wash in 1 ml 0.1 M trisodium citrate in 10% ethanol / 70% etOH...
tnx again...
Hello Nicola, I am trying to proceed DNA extraction from muscle using Trizol (Invitrogen). I saw your post up here, and I got interested how you solve your issue. Are you following the protocol from "Simultaneous isolation of DNA and RNA from the same cell population obtained by laser capture microdissection for genome and transcriptome profiling." How much gDNA did you get??? Thanks.
Thank you very much Andrea, for the detailed protocol, but I have a doubt: Gel Phase-lock tube? May use an ordinary tube?
I totally changed my protocol, Altheia...
I went back to the "classical" gDNA extraction as Olivier Kassel suggested.
The yield was higher than trizol procedure...
But Andrea Biloglav's protocol seems to be interesting...
I think ordinary tubes work well...
Also, I think I'll substitute 20 % glycogen with LPA...
Hello every one,
As Nicolas, during my Trizol dna extraction from tissu sample i met the same problem, very bad dna quality even if high quantity. Can someone please tell me if it is realy possible to extract dna from samples used to extract rna with Trizol, i conserved the organic phases for more than 1 year after rna extraction.
Thanks in advance.
Walid.
@ Walid Chemso, How did u solved the problem as I am facing the same situation? Can I use some kit for switching from the trizol method?
@ Saadiya Zia, the problem was not resolved, i had dna with lower quality..... I dont think thak there are kits to switch from the trizol method.
@Andrea, I have same question as Aletheia,
1) what is the maximum yield you got for DNA extraction using Trizol?
2) Could ordinary tube work instead of Gel Phase lock tube?
Thanks
@Andrea Biloglav , do you use PLG Heavy or PLG Light Gel Phase lock tubes?
I used method mentioned by Andrea (Only Day 1 method) for 4 samples. I got yield of 25 ng, 202 ng, 215 ng and 45 ng/ul (dissolved in 20ul 1xTE, pH 8.0)) with A260/280 ratio ranging from 1.45 to 1.60. Now, how to improve the quality of DNA? Is this DNA quality enough for routine PCRs?
I am using Back Extraction Buffer for isolating DNA from lung biopsies after I have isolated RNA using mirVana miRNA isolation kit. I am getting A260/280 ratio of 1.75-1.85 which I think is good for downstream PCR applications. However, A260/A230 ratios are usually very low ranging from 0.8 to 1.2 suggesting some chemical contamination. What can I do to improve A260/230 ratio? Any suggestions would be highly appreciated. Thanks.
Nicola Lo Buono
You can use this protocol as it worked well in my lab work.
TRIzol Reagent User Guide - Pub. no. MAN0001271 - Rev. A.0 (thermofisher.com) Sachin Kumar
Kafila Kousar
I'm a bit confused with this protocol. In the third part, just when you have to solubilize the DNA, it says: resuspend the pellet with NaOH 8 mM and then transfer the supernatant in a new tube after a centrifugation. Here my question, If I want to quantify the DNA in the samples, when I have to solubilize them in water? If you follow step 4, my samples are in NAOH 8mM, but to quantify I need my DNA solubilized in water...Could you help me with this question?
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