DNA can be damaged in a number of ways. Although the level or type of damage may not affect your experiments, sometimes enough damage can be sustained to ruin them.
Forearmed is fore-warned so here are 5 ways that your DNA can be damaged. Now you can do your best to ensure that any damage is minimised, giving your experiment the best chance of working.
1. Exposure to UV light. I have gone on about this so many times it is getting boring. But the fact remains that UV light cases thymine dimers and has wrecked many a good cloning experiment. So minimize you sample’s exposure to UV, especially the more damaging short-wave variety. Enough said.
2. Mechanical shearing. Excessive rough handling (e.g. pipetting or vortexing) of DNA can cause breaks and nicks. The longer the DNA, the more sensitive it is to shearing so treat things like gDNA especially carefully if you require intact DNA.
3. Phenol extraction. Phenol can oxidise bases, especially guanine. The oxo-guanine which can mis-pair with adenine, resulting in faulty replication and mutagenesis. Good job there’s hardly any need to do any phenol extraction any more!.
4. Dessication. Breaks and nicks, as well as base oxidation, can also be caused by harsh drying of DNA. This was shown under particularly harsh experimental condition, in which samples were dried rapidly in silica, but it is probably a good idea to avoid over-drying DNA anyway in general to prevent this sort of damage.
5. Heating. In aqueous solution, DNA is subject to oxidation and acid hydrolysis damage. Like most chemical reactions, these speed up as the temperature increases, which is why we store DNA at low temperatures and you should avoid excessively heating your DNA samples.
While most may think standard Taq is the backbone of PCR, there are many other DNA polymerase options out there. The polymerase you use has a significant impact on the efficacy of your PCR, specifically on the product yield, the purity of the product and the faithfulness with which the starting product is transcribed. Sometimes, […]
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