I plan to do some comparative quantitative studies on monoamines in insects, and I found that using DNA amount is a good way to normalize for differences in shape/size/cell number etc. that might be present among different species I'm looking at.
In the process of extracting the amines, I use acidified acetone, and homogenize the brains. I'm going to be left with cellular debris from the samples post-extraction of amines, from which I would like to quantify the DNA amount.
The DNA amount is meant to be used only as a normalizing factor. I have no other use for it.
I'd like to know what is the easiest/quickest way to do this. I already have a DNeasy extraction kit for Blood and Tissue, but in case I find a "DNeasier" method, or if there might be any problems with the kind of starting material I have, I'll be grateful.
Update May 2019:
I have tried the following extractions: phenol:choloroform:isoamyl alcohol, TRIzol and Tris/EDTA methods.
None of the methods work for the starting material, which is cell debris remaining post neurotransmitter extraction in a mixture of acetone, formic and ascorbic acid. Perhaps the solvent degrades DNA.
On the other hand, the Tris/EDTA method worked best for extracting DNA from 5 pooled heads of fruit flies. I have decided to go with that.
Uwe Wegner , since I haven't actually started the experiment, I haven't yet tested any particular method. Would you suggest that phenol/chloroform extraction for my particular purpose is a simpler/faster way than other ways to get quantifiable DNA?
I think it is an easy and cheap methode. Most of your cell debris, as well as proteins, should dissolve in the organic phase, while the DNA is located in the aqueous phase.
The commercial kits I know use columns to bind DNA, and you have always a loss of DNA during cleaning steps and elution.
So a simple protocol would be something like 25:24:1 phenol: chloroform: isoamyl alcohol mixture added to my homogenized and pelleted sample, vortex, spin down, and transfer the aqueous phase containing the DNA.
For the quantifying part, I suppose an ethanol precipitation, followed by drying and resuspension in nuclease free water is required? I could measure the concentration using a nanodrop or something? Or am I missing something else as well?
Sounds good.
You can measure the concentration with a nanodrop, but make three or more measurement per sample, because it is quite usual, that mistakes slip in during pipetting the sample onto the nanodrop.
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