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.
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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.