Laboratory 4k
Estimating the Amount of DNA in a
Sample Using Agarose Gels
Background
It is helpful to be able to estimate the amount of DNA in an experimental sample. Several
methods may be used to detect DNA and estimate its concentration or mass in a sample. If there
is enough sample volume, the DNA concentration may be estimated using UV spectrophotometry
(see in Chapter 7.) Indicator testing, such as that done with the NUCLEIC dotMETRIC™ kit,
allows for estimation of the mass of DNA in a sample.
Gel electrophoresis may also be used to estimate or quantify DNA. Running a few microliters
of a DNA on an agarose gel can give an estimate of the amount of DNA in a sample. If samples
of known concentrations (standards) are loaded onto a gel at a specified volume, then the mass of
DNA loaded in those samples will be known. Running unknown samples of the same volume on
agarose gels at the same time as the standard samples give a method of estimating the amounts
of DNA (mass and concentration) in the unknown samples by comparing the glowing in the
stained gel.
Purpose
To prepare a serial dilution of a known sample of DNA for use as a concentration standard.
What is the amount of DNA, measured in µg, in an unknown sample?
What is the smallest amount of DNA, measured in µg, visible (resolved) on a 0.8% agarose gel?
Materials
Microcentrifuge
Gel box, horizontal, for
agarose gels
Prepared agarose gel (from
Lab 4i)
Electrophoresis Buffer
concentrate, TAE (40X or 50X)
or LB buffer (10X or 20X)
Beakers, 600 mL
Tube rack for 1.7-mL tubes
Reaction tubes, 1.7 mL
Permanent lab marker pens
DNA samples (spooled
Lambda DNA (uncut), 500ng/
μL, 2 tubes for 16 lab groups
DNA loading dye, 10X (for TAE
gels) or 5X LB loading dye (for
LB gels)
Micropipet, P-10
Micropipet, P-100
Micropipet tips for P-10
Micropipet tips for P-100
Lambda/HindIII DNA sizing
standard markers, 50 ng/μL
(pre-mixed 90 μL sample + 10
μL of 10X DNA loading dye
for TAE gels or 20 µL of 5X LB
loading dye for LB gels
Power supply, 300V
Glasses, safety, plastic
Weigh boat, 5.5”X 5.5”
DNA gel stain (ethidium
bromide, 0.5 μg/mL or 1X
LabSafe Nucleic Acid Stain™)
Deionized water
Gel photo imaging system
Gloves, large
Procedure
Note: This activity assumes prior knowledge and experience preparing and running agarose gels.
Refer to Lab 4i and Lab 4j if you need help preparing electrophoresis buffer or agarose gels or
with loading, running, staining or imaging an agarose gel.
1. Prepare enough 1X electrophoresis buffer to prepare and run a gel. Record the calculations
and a diagram to show how you prepared the buffer. Are you using TAE buffer or LB buffer?
Find the correct DNA loading dye to use in step 4.
2. Prepare 0.8% agarose in 1X electrophoresis buffer with either 1 row of 8 wells or 2 rows of
6 wells. Pour a gel that is 6–7 mm thick. Leave to cool, undisturbed for 15 min.
3. Prepare four DNA samples at different concentrations to use as concentration standard