I test some DNA sample in qPCR and check the results.
And my colleagues said the DNA is degraded. What’s make DNA to degrade? I just know freeze and thaw too many times cause the degradation.
Can anyone tell me?
If possible, show me the reference thank you very much.
Many things can cause DNA degradation. As you pointed out if you freeze and thaw the DNA, the ice crystals that form will cut the DNA into pieces thus degrading it. You do not need to do this many times for the DNA to degrade. As few as one or two times will do it.
Other causes of DNA degradation include:
1. Contaminants in your DNA preparation. If your DNA was not purified well, some DNA degrading enzymes may remain in your solution which will degrade the DNA
2. Nucleases in your skin can cause DNA degradation, so handling DNA without gloves can cause DNA degradation. Some people lots of nucleases in their skin, others not as much.
3. If you store your DNA in a solution that is not buffered, or chelated, then your DNA will likely degrade. Make sure to keep your DNA in a buffer like TE, and at a pH of 8 or so to keep it stable
4. When handling DNA it is a good idea to keep it could (in an ice bucket or something like that). If you are keeping your DNA at room temperature for extended periods of time, while working with it, this can cause it to degrade
I am sure there are other reasons, but these are the ones I can think of right off my head. I short, if you do think you are getting degradation, then purify a new batch of DNA and aliquot it so that you do not have to freeze and thaw your DNA more than once or twice.