In order to be able to visualise DNA, one genotyping technique employs Phosphorus-32. This technique is called the Southern Blot. Phosphorus-32 is radioactive. Working with P-32 is a bit awkward, as one not only has to work in a fume hood designed for working with biohazardous material, but also behind a shield. Additionally, laboratories using P-32 must take care in disposing of all P-32 properly.
There are products available which claim to be as effective as P-32 in producing results. I had some experience with one such alternative a few years ago. Many weeks of attempting to troubleshoot the process yielded no useful results at all. Eventually, I gave up when I heard that others had similar difficulty with P-32 alternatives, so I had decided to stick to the radioactive method.
Another genotyping method in very common use in molecular biology is the Polymerase Chain Reaction. This method amplifies the DNA, and then the product is ran through an agarose gel containing Ethidium Bromide using electrophoresis. The EtBr allows the amplified DNA strands to be visualised under UV light.
EtBr is mutagenic, carcinogenic and tetratogenic. In a first year biology lab, only the teaching assistants were allowed to handle it, and they dealt with it in a fume hood. The students were not allowed to even be near the fume hood when the EtBr bottle was being opened!
Recently I was asked by a co-worker to test out a new alternative to EtBr, which is advertised as being safer. It also promised to give better results than EtBr. I thought to myself “Here we go again. It’s not going to work, you know, right?” Looking at the beautiful pictures included in the product pamphlet I wondered what wizadry was used to create these images.
I decided to divide the product of a PCR between two agarose gels. One agarose gel made with 5ul/100mL of EtBr, the other with 5ul (20000X)/100mL of the safe stuff. After placing 8ul of sample in each well, I ran the gel. Placing it on the UV light box, and switching on the light, I beheld the results:
So from now on, there will be one less mutagen in the lab. o/
Warning: The safe stuff can cause skin irritation and eye irritation.
– Ethydium Bromide: negligible really
– Safe Stuff: ~ $200 for 1mL, so enough for 200 100mL gels. (Shelf life of one year at room temperature, 2 years at 4C).
– Not having to expose oneself to EtBr daily: priceless