Now a days doing a DNA extraction for every experiment is quite common and after extraction the next step is to check the purity of the DNA which we do by doing a electrophoresis,and many other things are done such a cloning ,PCR amplification as well.So when we run it in a gel we use Ethidium bromide as an agent to view our sample ,but we know that this EtBr is a carcinogenic and mutagenic agent so what are the changes or the risk involved if we are using EtBr in our sample these are few question which arise while we do experiments
For example sometimes we do DNA cloning, and we do PCR as well ,the PCR amplicon is run in agarose and it is detected by ethidium bromide marking under UV light.next gel is sliced, DNA extracted from gel using an extraction kit like qiagen. until eventually you have your transgenic organism,while doing so, we use the very same DNA sample that was "contaminated" by ethidium bromide, so does this DNA you are using for transformation contain ethidium bromide? if so, how can it affect in numbers to mutation risk?
So what we can do to minimize the effect ?
Purified DNA contains negligible amounts of ethidium bromide. PCR and gel clean-up kits remove it quite well. There is, though, a risk of mutation from the fact that you're visualizing the gel in UV light with ethidium bromide. The risk of mutation from UV is minimized by exposing the gel as little as possible, and by using "preparative" transilluminators that come with a low-light setting for cloning purposes. If you work well and quickly, the chance of mutagenesis isn't higher than the chance of a polymerase incorporating the wrong nucleotide.
For example sometimes we do DNA cloning, and we do PCR as well ,the PCR amplicon is run in agarose and it is detected by ethidium bromide marking under UV light.next gel is sliced, DNA extracted from gel using an extraction kit like qiagen. until eventually you have your transgenic organism,while doing so, we use the very same DNA sample that was "contaminated" by ethidium bromide, so does this DNA you are using for transformation contain ethidium bromide? if so, how can it affect in numbers to mutation risk?
So what we can do to minimize the effect ?
Purified DNA contains negligible amounts of ethidium bromide. PCR and gel clean-up kits remove it quite well. There is, though, a risk of mutation from the fact that you're visualizing the gel in UV light with ethidium bromide. The risk of mutation from UV is minimized by exposing the gel as little as possible, and by using "preparative" transilluminators that come with a low-light setting for cloning purposes. If you work well and quickly, the chance of mutagenesis isn't higher than the chance of a polymerase incorporating the wrong nucleotide.
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