Genetic Engineering
Fall 2011
Is it DNA? Nucleic acid measurement using A260/280
Now that you have a sample of DNA we have to use techniques for measuring how
much DNA, and for allowing to see the DNA, albeit indirectly.
Samples of nucleic acids are often evaluated for purity by a procedure commonly
known as the A260/280 ratio. Why “purity?” In the extraction procedure it’s possible that,
along with the DNA, you also collected some protein or some RNA. Too much of either can
interfere with other procedures in which you might use your DNA sample. The A260/280
procedure is not a definitive test of purity, but it is quick and does give a reasonable
assessment of the sample – both the purity and the quantity. The amount of light (of  =
260 nm) absorbed by DNA is directly proportional to the quantity of DNA in the sample
A portion of the sample of purified nucleic acid is placed in a UV spectrophotometer.
The absorbance of the sample is measured at 260 and 280 nm, and a ratio of the
absorbances is made. Pure DNA has an A260/280 or 1.8, and pure RNA has an A260/280 of 2.0.
Protocol for measuring the purity of a DNA preparation:
 Add 990 L of deionized water to a fresh cuvette, then add 10L of your sample –
mix gently with the pipetter

Put your cuvette in the spectrophotometer and “read” the A260/280

Construct the A260/280 by first subtracting the blank reading at 260 nm from the
sample reading at 260, for each individual well. Repeat for 280 nm. Use these
adjusted absorbance values to construct the A260/280 for each sample
o Samples with very low DNA concentrations may not yield accurate ratios,
and samples with extreme protein contamination may also give a ratio far
from 1.8
Calculating the concentration of DNA in your sample
 1 O.D. A260 unit of dS DNA = 50 µg/ml

When calculating the conc. of your sample, adjust for any dilution you made when
measuring the A260 of your sample

A260 of your sample X 50 = ug DNA/ml of your sample X dilution
Can your see your DNA sample? Electrophoresis of nucleic acids
In today's lab we will also learn the technique of separating nucleic acids using
agarose gels and an electric current; or, in other words, by electrophoresis. In general
terms, electrophoresis is the process of migration of charged molecules through solutions
in an electric field.
In our application, the molecules are the nucleic acids (we'll use both your DNA and
RNA preparations), and the solution through which they migrate is a buffer-filled matrix of
agarose polymers.
DNA or RNA samples are loaded into wells of an 0.8% agarose gel. (Agarose is a
polysaccharide that forms long chains). An electrical field applied across the gel causes the
nucleic acids to move from their origin (the wells) through the gel matrix towards the
positive electrode. The gel matrix acts like a sieve through which smaller molecules migrate
faster than larger ones.
In many instances the gel will be stained with ethidium bromide after the samples
have been separated sufficiently. Because the nucleic acids are not visible in the gel, a
tracking dye is added to the samples so that the progress of the samples may be monitored
indirectly (you do not want your samples to run out of the gel, which they will do if left
exposed to the electric field long enough).
In this lab we will add a non-toxic DNA stain to our samples prior to loading them on
the gel. We should be able to see the DNA right away, but the stain may not work that well
with RNA.
Run your samples on your gel, proceed carefully through the following steps:
Making the gel solution
 Make 35 ml of 1% agarose in SB buffer (supplied by me) in a 125 ml flask
 Heat at 15 second intervals until all agarose is dissolved - careful, the flask will be
hot
Preparing the sample
 We want about 0.5 ug of DNA per lane:
o add 1 ul of gel loading dye to a volume of sample that will give about the
right amount of DNA
o add 1 ul of GelStar nucleic acid stain
 Run 2 or 3 lanes of each sample
Loading the gel
 Use a pipetter to load your samples and standard into the wells of the gel
Running the gel
 Set power supply to 75 volts and start the gel running
 Let the gel run for about 15 minutes
Visualizing the DNA
 We will use the digital camera, light box, and computer to capture an image of the
DNA in the gel