Hi. I'm testing the existence of parasites on the surface of commercial vegetables by using real-time PCR method.
I extracted genomic DNA of vegetable samples using QIAamp stool mini kit.
Before extracting DNA, I washed my vegetable samples with 0.1% liquinox and PBS, and centrifuged twice to get the supernatants.
I stored my supernatants in -80C before extracting DNA.
After DNA extraction, I measured A260 and A280 to quantify the density of DNA. But the density of DNA was nearly same as BLANK.
Protocol says, test samples must be stored at 4C or -20C
I suspect the storing condition is the major problem.
But I don't understand if -80C can damage genomic DNA and degrade them.
Can anybody explain this phenomenon? Thank you for everybody.
Hi, maybe it could be your extraction technique and method. Try to optimize the protocol and improve your technique. The problem should be solved because DNA is even stable at room temperature and I have kept mushroom and plant samples in -80 before, the DNA yield and quality are both good.
What are these pathogens? Microbes, nematodes, fungi, etc? I'd choose a DNA extraction kit to match the organism. Are you sure that these samples actually have the pathogens?
I'd first increase the amount of starting materials and choose a proper kit. Don't just use whatever is available without verifying that it will work well for your organism.
-80C freezing does not damage gDNA.
I would suggest you to be more sterile due to the pathogens. Moreover, you can also use some homogenization methods like physical one with some machines or with liquid nitrogen.
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