Does anyone have experiences in treatment of samples (from food or pure culture) by DNAse to digest free DNA prior to DNA-extraction? The free DNA shall not be amplified by real-time PCR and will therefore be eliminated by the DNAse? DNA of intact cells should be protected by their cell wall.
Does anyone have
experience concerning success
a protocol? (DNAse concentration, time of incubation, reagent buffer, inactivation of DNAse)
Many thanks in advance!
The ammounts of free DNA in cell cultures is extremly low, compared to gDNA and does not influence susequent real time PCR.
Do you need to remove RNA as well, or is DNA your only concern? The recommandations will depend on many factors, like cell types, volume, etc. There used to be kits available for single cell treatment, maybe also larger volumes. You will need a DNase that is easy to inactivate or remove.
Unfortunately, I am not into details here. I suggest you contact Cathrine, [email protected] to discuss the case.
There is also a poster on the subject: Aune, Marthe; Olsen, Gunn-Hege; Elde, Morten; Henriksen, Jørn H.; Berg, Thomas: "DE-ICE: Differential Extraction Improved by a Cold adapted Enzyme (HL-dsDNase)." Human Identification Solutions Conference, Madrid 2015; 2015-03-03 - 2015-03-04
This might be true, but we are analyzing food samples and detect DNA from microorganisms, that might have been damaged or killed during the production process
DNAaseI may appear to be too expensive for these purposes. I think, that prior DNA extraction, you may treat your samples with hydrogen peroxide. It destroys DNA and washes it away, leaving the cells intact.
Hi,
If your goal is to identify bacteria in food or pure culture try to use kit dedicated to isolating DNA from bacteria, for example, Genomic Micro AX Bacteria Gravity (Gravity flow kit for genomic DNA purification from bacteria, 0517), A&A Biotechnology.
You can have positive results from plant or animals (food of animal origin) DNA if you use the kit for isolated total genomic DNA.
Next problem can be not specific primers. Try to design new more specific primers.
Good luck!
Hi Sabrina,
Have you heard of the qPCR-PMA method? or propidium monoazide (PMA)?
I think it could be a good choice for your free DNA problem.
I send you the link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549832/.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology