NAME: ________________________________
PREPARING DNA SAMPLES AND RUNNING A GEL
PURPOSE
To learn the
technique.
Standards Addressed
gel
electrophoresis
INTRODUCTION
DNA can be analyzed using the gel
electrophoresis technique. When an
electric field is applied across a gel,
negatively charged DNA molecules will
migrate through the gel matrix toward
the positively charged cathode at a rate
relative to size. An analyst determines
the size of DNA fragments by the
migration distance; smaller fragments
will move farther through the gel than
larger fragments. Before the DNA can
be loaded onto the gel it first needs to
be mixed with a loading dye. When
current is applied, not only does the
DNA move but so does the loading dye.
Within five to ten minutes two or more
distinct bands appear. These are
markers that show the progress of the
DNA down the gel. After the gel has
been allowed to run it is then stained
with a dye that adheres to the DNA due
to its negative charge. Next, the DNA is
ready to be put in a de-staining
solution to remove excess dye. When
the de-staining process is complete it is
now time to view the gel. Distinct
bands should appear when the gel is
contrasted against a light source
usually a gel lamp. At this point the
DNA bands are ready to be measured
and analyzed.
Now let’s try to run a gel!
Standard 1: Science As Inquiry
Benchmark 1:
The student will demonstrate the
abilities necessary to do scientific
inquiry.
Indicator 3:
The student correctly uses the
appropriate technological tools
and mathematics in their own
scientific investigations.
Standard 3: Life Science
The student will develop an
understanding of the cell,
molecular basis of heredity,
biological evolution,
interdependence of organisms,
matter, energy, and organization in
living systems, and the behavior of
organisms.
Benchmark 1:
The student will demonstrate
an understanding of the
structure and function of the
cell
Indicator 3:
The student understands
cells function and replicate as
a result of information stored
in deoxyribonucleic acid
(DNA) and ribonucleic acid
(RNA) molecules.
Benchmark 2:
The student will demonstrate
an understanding of
chromosomes, genes, and the
molecular basis of heredity.
Indicator 1:
The student understands
living organisms contain DNA
or RNA as their genetic
material, which provides the
instructions that specify the
characteristics of organisms.
MATERIALS
Latex Gloves
Safety Glasses
Loading Dye
Gel Box
Microtube Rack
Microtubes
Nucleostain
Gel Lamp
1 Microtube Crime Scene DNA
4 Microtubes of Suspect DNA
0.5-10 uL Micropipetter
0.5-10 μL Micropipetter Tips
Container for Tip Disposal
λ DNA Ladder
1 X TAE Buffer
PROCEDURE
1. Before beginning, be sure to glove your hands and don safety glasses.
2. Have a properly prepared gel box as described in earlier protocols.
Diagram and label a properly assembled gel box.
Do not forget to include the power source and appropriate connections.
3. In a microtube rack place:
Sample DNA
Microtubes containing suspect DNA
Microtubes containing crime scene DNA
Microtubes containing DNA ladder
Microtubes containing Loading Dye
6 empty microtubes
4. Using a permanent marker label 5 microtubes as
follows: suspect A, suspect B, suspect C, suspect
D, and crime scene. Label the microtube
containing the DNA ladder as well.
To save
time and
tips,
it is not
imperative
to change
tips
between
tubes at
this point.
Explain why it is necessary to
label all microtubes.
5. Using a 0.5-10 uL micropipetter with a 0.5-10 μL micropipetter tip place 5 uL of loading
dye in each of the five tubes marked for DNA. In the microtube containing the ladder add
only 3 uL of loading dye.
The loading dye is actually two dyes with distinct molecular weights.
Why is it advantageous for the dye to be composed of such a mixture?
6. Using a fresh tip, transfer 10 uL of suspect A DNA into the corresponding
microtube containing the loading dye. Repeat this step with the remaining 4
suspect DNA samples.
Be sure to change tips between each suspect sample. We don’t want to mix DNA!
Helpful Hint: When adding the DNA to the loading dye it is
important to mix the two solutions together. This can be
achieved by pipetting the combined solution up and down 3
times.
7. Using a fresh tip load 3 uL of λ DNA ladder into the
tube marked for the ladder. Be sure to mix the
DNA with the loading dye by using the above
technique.
The ladder is used as a distance
measurement in relation to the
size of the DNA pieces in the gel.
This ladder is a way to reference
how each gel ran because no two
gels run exactly the same.
Calculations and statistical
analysis are used to compare
different gels to one another.
Predict the relative distances the
varying lengths of DNA fragments
will run. Explain your reasoning.
Stop and read step 8 carefully!
8. Loading a well is a very delicate process. Follow the
steps described below to ensure the wells are not
damage during loading.
 Pull up the solution you wish to load and begin
transferring into the well by slowly and
carefully placing the tip inside the well.
 Feel for the edges of the gel gently by moving
the tip back and forth.
 Do not poke the pipette tip into the bottom or
the sides of the gel.
 Slowly expel the DNA/loading dye solution into
the well. Press the pipette button only to the
first stop. There will be solution remaining in
the tip.
The second stop on a micropipetter expels a burst
of air which serves to push the last drop of liquid
out of a tip. This burst of air is strong enough to
break a well. For a successful gel it is not
important to expel this last drop of solution.

Slowly pull the pipette tip out of the well but do
not release the plunger. This can cause the
sample to be sucked back into the pipette tip.
Be sure to change tips between each suspect sample. We don’t want to mix DNA!
9. Using a 0.5-10 uL micropipetter with a 0.5-10 μL
micropipetter tip, load 6 uL of the ladder-dye mix
into the first well.
Remember to change tips between each sample.
10. Load 10 μL of the DNA samples in the next 5 wells.
Remember to change tips between each sample.
11. Place the top of the gel box onto the gel tray. Make
sure that negative (black) leads connect with
negative and positive (red) leads connect with
positive.
The gel should be run as soon as possible.
Offer a reason as to why this is necessary.
12. Plug the box into a power supply by matching the negative plug with the negative
input slot and the positive plug with the positive input slot.
13. Set the Volts at 75 V, set the Amps to 185 A and set the time for 90 minutes. Make
sure that the K button (second button down) on the box is reading for Volts and not
Amps. After checking the settings again, press the run button.
Helpful Hint: if everything is working correctly, little bubbles from
the electrodes should appear in the well.
14. After a few minutes check to see if the DNA sample is running from Negative (black
electrode) to Positive (red electrode). Distinct blue and purple bands should appear
within 5 to 10 minutes. This is the loading dye and can be used as a marker to
follow the progression of the DNA.
15. When the gel is finished running, turn the power
supply off and remove the lid from the gel box.
16. Gently take the gel out of the box and place it in a
plastic container. Add Nucleostain until the gel is
completely submerged. Place a lid over the
container and let the gel soak overnight.
What is the purpose of staining the gel?
17. Once the gel has been allowed to stain overnight
put on gloves and remove the gel from the staining
container. Place the gel in an empty container
and add 1 X TAE buffer until the gel is submerged.
On a molecular level, explain why
the gel can be destained while the
DNA maintains its stain.
Helpful Hint: Nucleostain may be poured back into
its original bottle and reused.
18. Remove the gel from the plastic container and
place it on the gel lamp. Now the gel may be
analyzed.
19. The distance that each DNA fragment moves is
used to calculate the Rf factor, an abbreviation
for “Relative Mobility of the Fragment”. The Rf
value can then be used to calculate the
number of base pairs in the fragment.
Calculate the Rf factor for each fragment using
the equation:
Rf = distance in mm that the DNA fragment migrated
distance in mm from the well to the dye
Graph the class’s data for analysis. Include
appropriate labels.
QUESTIONS AND CONCLUSIONS
1. What special property of DNA allows for gel electrophoresis and staining adhesion?
2. Describe two precautions you take when preparing and running a gel?
3. Why is a ladder loaded onto a gel?
4. After examining your graph, what conclusions can you draw regarding the size of the
class’s DNA fragments? What can be concluded regarding the restriction
enzymes used and the cutting process?
5. In addition to DNA analysis, name at least two other applications in which gel
electrophoresis could prove helpful.