I'm extracting DNA from nematodes using the Qiagen DNeasy Blood & Tissue kit but I'm struggling to get a high enough DNA concentration. I followed the manufacturer's instructions except I halved the amount of Proteinase K and Buffer ATL to lyse DNA because of how small the parasites are.
The parasites have been kept in 70% ethanol and have been grouped by morphology by a master's student. After researching extraction methods, I was going to next prepare the parasites by cleaning them with PBS buffer and grind them. Is this the best way to improve my DNA concentrations?
I have only been using one parasite per extraction as although they have been grouped by morphology, I am uncertain whether they are genetically the same. Would it be worth using more than one parasite per extraction to increase the DNA concentration? Should I confirm morphology under a microscope to use as many as possible?
The parasites are also a year or so old and some have decomposed, so this is also something that could potentially be causing low DNA concentration.
You can elute in a smaller volume of buffer. Heat the elution buffer to 65*C, let it sit on the column for 1 minute, then spin.
Or, to increase concentration you can always evaporate down the final volume by putting the tube with the lid open in a warm bead bath.
One nematode isn't going to yield much DNA to start with, so most folks do pool them together unless you suspect that they are in fact NOT the same.
Another tip is that you can do repeated freeze-thaw cycles (-80*C freezer to room temp) to help rupture the cuticle.
Good luck!
I would use phenl/chloroform extraction followed by adding a co-precipitant like glycogen to increase predipitation. Column methods are very inefficient at generating pure dna and the losses are great. Consider using nested pcr to get the most out of a tiny amount of dna
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