Laboratory 4d EtBr Dot Test: A Quick Test for DNA in Samples Note: Some states prohibit the use of ethidium bromide in college teaching or high school laboratories. As an alternative to the EtBr Dot Test, use an DNA indicator kit from G-Biosciences called NUCLEIC dotMETRIC™ which is available for purchase at www.gbiosciences.com/ResearchProducts/BTSNMSupport_Materials.aspx. Background To test for the presence of DNA in a solution, a quick ethidium bromide (EtBr) test can be conducted in a matter of moments. An EtBr test indicates the presence of DNA by glowing a “hot” pink-orange color under ultraviolet (UV) light. When EtBr is mixed with DNA in solution, the EtBr intercalates (fits between) between the nitrogenous bases of the DNA molecule. This causes the bases to move farther apart and interact with light in a different manner. Shining a UV light on a mixture of EtBr and DNA will reveal a glowing pink-orange color. The degree of “glowing” indicates the presence and amount of DNA (see Figure 4.5). Since EtBr can change the shape of DNA molecules, it is a hazard to human cells. It is a known mutagen (can cause changes in the DNA code) and is a suspected carcinogen (cancer-causing agent). Safety precautions, including wearing goggles and gloves, must be taken when using EtBr or when working in an area where EtBr is used. Purpose What are positive and negative tests using EtBr as an indicator? Do the DNA samples test positive for DNA using the EtBr dot test? Materials Gel photo imaging system Paper, thermal Printer, thermal DNA, salmon testes, 2 mg/mL DNA samples Gloves, large Glasses, safety, plastic Ethidium bromide, 0.5 µg/mL Micropipet, P-10 Micropipet tips for P-10 Deionized water Safety Precautions • EtBr is a hazardous chemical. • EtBr is to be prepared and used only by the supervisor. • Wear goggles and gloves while in an area where EtBr is used. 74 Chapter 4 Laboratory Manual protective samples and controls mixed 1:2 with EtBr shield + control sample A sample C sample B sample D – control on Transilluminator results: A=3 B=4 C=2 D=0 off Relative Amount of DNA Glowing as Compared to Controls Key 5 0 5 = as much as + control 3 = half as much as + control 0 = no glowing with EtBr, no DNA Figure 4.5. EtBr Dot Test. When EtBr is mixed with DNA, the mixture glows under UV light. When the sample is compared with the positive and negative standards, one can determine the presence of DNA.