I isolated some species of bacteria from oral cavity containing a gene (secret). I detected the gene by PCR in the genomic DNA obtained from broth cultures. I stored the broth cultures in 20%v/v glycerol in -80C freezer. After a few weeks, I grew the bacteria from stock on fresh agar plates and prepare the genomic DNA again. However, this time I could not detect the gene in the newly prepared genomic DNA from the same species. Could anyone please help me to detect the cause of the loss of this gene?
I don't think that these storage conditions can be the cause. I assume that the DNA isolation method was the same before and after preservation? Errors in the PCR setup can be excluded (e. g. enzyme/primer problems, inhibitors)?
Hi,
The loss of gene is possible...but all the colonies may not loose the gene at the same time....You need to include proper controls to arrive at such conclusions. As mentioned above the bad quality DNA preparations can result into no amplification....also any PCR reagent may also have gone bad....so proper controls are must....repeat the experiments..
I think this applies for most methods: If there is complete, absolute signal loss, it's most likely technical problem. If technical problems can be excluded with 100% confidence, you've found something important
If a bacterium loosing genes from genome it should be by deletions. But i never observed that temperature affect for gene loss and we store plasmids transformed E. coli clones in glycerol at -86 C. The problem should be in PCR or DNA extraction procedure.
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