I perform total RNA isolation according to RNAzol RT protocol from bone marrow samples, but I need the DNA of this samples too. Some protocols explain that I can use DNAzol to isolate the DNA but this is not soluble in water or TE, only in 8 mM NaOH. But I would not want to use this solution because i have to use HEPES after to adjust pH of DNA solution.
If you use Trizol, you can separate the DNA and RNA in the same sample. If you need to DNA you stop in the initial step, or go ahead you'll have RNA.
Neutralising the NaOH is not a big problem, but dissolving DNA in NaOH or water will make it single-stranded, and that might be important, depending on what you want to do with it.
I would ignore the protocol and try dissolding the DNA from the DNAzol method in standard TE (10mM Tris pH 7-8 1mM EDTA), increaseing he temperature to 50-60Celcius for perhaps 30 mins. Centrifuge and check DNA concentration on nanodrop. Then vortex and continue heating until the E260 no longer increases.
Dear Geoff, thanks... I will do your recommendation, and also I will perform a simple DNA phenol extraction to avoid insolubility of DNA.
Just to note that DNA isnt insoluble in neutral aqueous buffers, unless is has been precipitated with something like cetyltrimethylammonium bromide. And if it has tightly bound histone proteins, for example, it might take longer to dissolve, but dissolve it will...eventually. There are numerous mentions on these forums about "overdried" DNA. This is just folklore, in my experience.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA