Princeton Molecular Biology Outreach Programs @ Springside School

The goal of the experiments chosen for this Workshop is to illustrate the power of state-of-the-art
molecular biological techniques. In an increasing number of newspaper and journal articles, websites, etc.
about The Human Genome Project and other scientific advances, it should be clear that genes and genetics
have become major topics of discussion in society today.

All of genetics stems from the sequence of bases along the double helical DNA molecule. In addition
to the obvious traits that are most frequently associated with genetics, such as eye and hair color, an
increasing number of specific tests can detect precise sequences within regions of DNA molecules that
have enormous implications for health and legal status. For example, accurate tests have been developed
for the detection of numerous genetic disorders such as cystic fibrosis, sickle cell anemia and many others.
In addition, analysis of the human genome has shown that different human genomes are 99.9% identical
and that the divergent 0.1% is comprised mostly of point mutations known as single-nucleotide
polymorphisms (SNPs). This discovery has led to the method of haplotype mapping, which correlates a
specific pattern of SNPs in a particular chromosomal region with susceptibility to a certain genetic
disorder, such as diabetes or age-related macular degeneration (AMD), the leading cause of blindness in
people over 50. DNA analysis has also become very common in forensics. It is now possible to determine
whether tissue samples as small as a single hair follicle or a smear of blood came from a certain individual
or suspect, as you will learn during the Workshop. In addition, conservation groups now use forensics to
combat illegal trade in protected animal species by identifying products that contain ingredients from rare
species. Also, analysis of DNA obtained from a community of microbes (metagenomic DNA) is used to
estimate the microbial diversity of natural environments of several niches in the human body.

All this information requires a detailed understanding of DNA, gene structure, and some sophisticated
technologies that have been developed only within the last 5 - 30 years. Yet, it is also critical to understand
the limits of these technologies, which often determine the accuracy of the tests as well as their ethical
implications. Some important questions are: Should an individual’s genetic information be private? How
much should you know about your genetic makeup? How much should others be allowed to know about
your genetic makeup? How should this information be used ethically? A thorough discussion of these
ethical questions is obviously beyond the scope of this Workshop. However, the design of the experiments
you will perform in the next will provide you with a basis for understanding the relationship between DNA
structure, modern genetics, and several social and ethical concerns.

In the laboratory, you should work with good laboratory technique at all times and keep your bench
area clean and neat after each experiment by always:

1. Cleaning all lab equipment, including parts of your gel apparatus, and returning these to the proper
places for storage.

2. Wiping your bench area using a paper towel and water.

3. Washing your hands when finished.

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Basic Microchemical Techniques: Agarose Gel Electrophoresis
& Micropipetting
I. Introduction: The analysis of DNA, deoxyribonucleic acid, frequently requires determining the size
of individual DNA molecules or even the nucleotide sequences of specific regions within DNA molecules.
Under most conditions, DNA has a relatively uniform acidic or negative charge caused by the phosphate
groups on the outer surface of the molecule. When placed in an electric field in a process known as
electrophoresis, negatively charged DNA molecules will migrate toward the positive electrode. In a
semisolid matrix like an agarose gel, larger DNA molecules will be retarded by the sieving properties of
the matrix, so they move more slowly and migrate a shorter distance in a given time than smaller DNA
molecules.

Today, you will first pour an agarose gel and then use this gel for electrophoresis of several different
DNA samples. After staining and photographing the resulting pattern of bands in the gel, you will use
your photograph to analyze these DNA samples by determining the sizes of unknown DNA molecules.
For this analysis, you will use two samples of DNA standards that contain molecules of known sizes to
plot a calibration or standard curve. Some of the samples you will analyze have been digested with
restriction enzymes, which recognize, bind to and cleave DNA at specific nucleotide sequences.
Approximately 300 different restriction enzymes are currently commercially available, and these enzymes
are very powerful tools currently available for analyzing DNA, as you have learned from your reading
and will demonstrate to yourselves during this Workshop.

In addition to electrophoresis, most of the procedures in this Workshop and also in research
laboratories employ microchemical techniques; i.e., using very small amounts of reagents such as DNA
and enzymes. Many reactions require the addition of only one microliter (1 µl, one one-millionth of a
liter) of some components, so it is essential that you become familiar with measuring small volumes
accurately and reproducibly. In addition, because many basic procedures in molecular biology use
bacterial strains, particularly Escherichia coli (E. coli) a superstar in molecular biology, you will also
become familiar with some basic techniques for working with microbes.

http://www.fao.org/docrep/005/AC802E/ac802e05.htm

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 GEL ELECTROPHORESIS & MICROPIPETTING
I. Micropipetting: Micropipettors are precisely calibrated > The P20 is used to transfer
volumes between 1 and 20 µl,
instruments that combine the functions of a pipette and a pump.
and the P200 is used for
Disposable plastic tips make micropipettors reusable. To measure the volumes between 20 and 200µl.
small volumes used in molecular biology accurately, you will use the It is preferable to use the P20 for
P20 and P200 Gilson adjustable micropipettors. volumes less than 20 µl, because
the P20 measures small volumes
The pushbutton at the top of the micropipettor has two working more accurately than the P200.
positions in addition to the fully upright position. The first position
is used for picking up liquid, and the second position is used to > The P20 should NEVER be
eject or deliver liquid from the tip. used for volumes greater than
20 µl nor the P200 for volumes
greater than 200 µl because
this will damage the pipettes.

1. To reach the first position, push the button down using relatively > If using your index finger is
light pressure with your index finger until you meet some uncomfortable, your thumb can
be used instead.
resistance.

2. Reaching the second position requires significantly more pressure

from your finger, so push the button down with more force to
reach this position.

3. Change the volume setting of the scale on the front of the P20 to > The two black numerals
read “1-0-0” from top to bottom using the black gears near the represent integer microliters,
and the red numeral represents
top of the pipettor. What volume does this represent? tenths of microliters. The
reading “1-0-0” indicates 10 µl
on the P20 as shown below:
Black Numerals
1
0
0 Red Numeral

4. Everyone should practice pipetting using your P20 micropipettor, > The white tip has graduations at
the white graduated tips and the red liquid as described next. 10, 50 and 100 µl, with the mark
nearest the point of the tip being
the 10 µl mark.
5. Put a white graduated tip firmly onto the end of the pipettor by > Using the pipettor to pick up tips
gently pushing the barrel of the pipettor into a tip in the rack. is preferable to picking up a tip
with your hand to avoid
contaminating the tip with
microbes on your hand. Then use
your fingers to push the tip gently
but firmly onto the barrel of the
pipette without touching the end of
the tip.

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6. Depress the button at the top of the pipettor until you reach the first > Be sure not to push the button
stop position and feel considerable resistance. down to the second stop position
when picking up liquid or your
measurement will be grossly
incorrect.

7. Keep the button at this position and insert the tip into the red > Do not insert the tip too far into
solution. the solution or extra drops will
cling to the tip and make your
measurement inaccurate.

8. With the tip still in the red liquid, SLOWLY release the pressure of > If you release the button too
your finger and allow the button to return to a full upright position. quickly, air bubbles could enter
the tip, and the volume of liquid
The red solution will enter the tip. picked up will not be correct.
Check the graduations on the tip
to determine whether the volume
you have picked up is correct.
9. Place the bottom of the tip just at or just above the meniscus of the
solution to which you are transferring (or near the bottom of a tube
if the tube is empty).

10. Depress the button all the way to the second stop position until all
the red liquid is expelled.

11. Use new graduated tips to transfer 5 µl and 1 µl of the red > Observe the amounts of liquid in
solution to the 1.5 ml microtube. the tip for these measurements and
try to remember how full the tip
appears for these volumes. If you
12. Continue practicing with the red solution and a microtube until know approximately how much
you can pipette accurately and easily. liquid should be in the tip, you
will recognize when your pipettor
misfunctions while you are
pipetting instead of after an
experiment fails.
13. Always use a new tip for each transfer because residual liquid on
or in the tip from a previous transfer could contaminate the new
solution or cause inaccurate measurements.

14. Micropipettors are very precise instruments that are expensive to

purchase (more than $280 each), repair and recalibrate. There are
three maneuvers that you should NEVER try with these pipettors:
a. Never measure a volume greater than the maximum
volume of a micropipettor, the volume indicated on the
pushbutton.

b. Never use the micropipettor without a tip. > Liquid will clog the pipette and
ruin parts.

c. Never lay down a pipettor with a tip containing liquid > The liquid could run back into the
attached. pipette and damage it.

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 Micropipetting Small Volumes: Each person should use the white > These exercises were adapted
graduated tips to perform the exercises described below. from Laboratory #1 of DNA
Science, pp. 327 – 328.

1. Use a black marker to label three clear 1.5 ml microtubes with the > The microtubes are in the whitish
letter “A”, “B”, or “C”. rack at your bench.

2. Set your P20 micropipettor to 5 µl and add 5 µl of Solution I (blue) > Be sure you use a new tip each
time you pipette. Use the chart
to each microtube, as shown in the chart below. below as a checklist to ensure that
you add the correct amounts to the
reaction tubes.
Tube Solution Solution Solution III Solution IV Total
I (Blue) II (Red) (Green) (Yellow) Volume
A 5 µl 4 µl 1 µl --
B 5 µl 4 µl 1 µl --
C 5 µl 4 µl 1 µl 5 µl

3. Use new tips to add 4 µl of Solution II (red) to each reaction tube.

4. Use new tips again to add the indicated volumes of Solutions III
(green) and IV (yellow) to the reaction tubes.

5. Close the tops of the tubes and mix the reagents by flicking each > Be sure there is a tube across the
tube with your fingers. rotor from each of your tubes for
balance. To operate the microfuge,
screw or press the top of the rotor
6. To bring the contents to the bottom of each tube, place your tubes on, close the lid, and spin the tubes
and your partner’s tubes in a microfuge and pulse spin them for a for three seconds. Centrifuging for
few seconds as described to the right. short times is accomplished easily
by holding down the button on the
front panel of the microfuge for
7. Remove the tubes from the microfuge. the required time and then
releasing it to terminate the spin.
8. Label tube “A” with your initials and place it in the rack on the
able at the front of the lab.

9. Check the volumes in tubes “B” and “C” yourself as described to > Check the volumes by setting your
the right. pipette to the total volume that
should be present in the first tube.
Then put a new tip on your
10. If you have pipetted accurately, the tip should be completely filled pipettor, depress the pushbutton to
with liquid; there should be no air space at the end of the tip, and the first stop, put the tip to the
no liquid should be left in the tube. Be sure to check the volumes bottom of the tube and release the
carefully because it is essential that you pipette accurately. button slowly so all the liquid in
the tube enters the tip. The tip
should be completely full if the
volume is correct. Repeat this for
your other tubes.
11. Discard tubes “B” and “C before proceeding.

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Micropipetting Large Volumes: Each person will each now practice
pipetting using his/her P200 pipettor.

1. Use a black marker to label three new (1.5 ml) microtubes “D”, > These tubes are in the whitish rack
“E”, or “F”. at your bench.

2. Pipette the volumes shown in the chart below into the appropriate > Use a new tip for each transfer.
tubes:
Tube Solution Solution Solution Solution Total
I II III IV Volume
D 20 µl 40 µl 50 µl 30 µl
E 20 µl 40 µl 50 µl 30 µl
F 20 µl 40 µl 50 µl 90 µl

3. Close the tops of the tubes and mix the reagents by flicking each
tube with your finger.
4. Spin the contents to the bottoms of the tubes for 3 sec in the
microfuge.
5. Mark tube “D” with your initials and place it in a rack on the front
table to be checked for accuracy.
6. Next, calculate what the volumes of tubes “E” and “F” should be
and check the volumes in these tubes yourself to assess your
accuracy.
7. If your volumes are not correct, be sure to ask a member of the lab
staff for assistance.

8. Discard tubes “E” and “F” before proceeding.

waynesword.palomar.edu/lmexer3b.htm

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III. Pouring an Agarose Gel: Agarose is a purified form of agar,
a complex polysaccharide derived from seaweed. When boiled in Electrophoresis is the process by
buffer, agarose dissolves and then solidifies upon cooling much like > which molecules are sorted by size
Jell-O. Agarose gels for electrophoresis are prepared in a pH- iin a semi-solid matrix like agarose
iin an electric field.
buffered solution, which conducts an electric current through the gel The phosphate groups on the outside
and provides the electric field through which the DNA molecules move. oof DNA give it a uniformly
nnegative charge, so DNA migrates
tttoward the positive electrode.
1. Prepare a 1% agarose gel (gel bed volume = 50 ml) by adding .5 g
agarose powder to 49.5 ml of 1X electrophoresis buffer.

2. Wearing gloves and working with your partner, place a black dam in > The dams are asymmetric. The side
each of the two slots at the ends of the gel bed. of each dam that forms a right angle
with the bottom of the dam should f
fface the gel bed as shown below to
tthe left.

3. Obtain a flask of molten agarose from the water bath. > Molten o1.0% agarose can be kept
iin a a65 C water bath so it will not
solidify.
4. Use the orange Hot Hand mitts available beside the waterbath to
hold the flask.

5. Close the cap of the flask and swirl the flask to mix the agarose.

6. Use a transfer pipette to seal the ends of the gel bed as shown below > Although the gel apparatus does not
with a narrow bead of agarose before pouring your gel. lbreak often, it is best to be certain
no leaks ever occur by sealing the
gel bed.
Dam Dam Do not overfill or underfill the gel
Seal with Agarose bbed or your gel will be too thick or
ttoo thin. Surface tension will make
the gel appear thicker than it is.
Observe the gel level closely while
Gel Bed ppouring.

7. After waiting one minute (min) while the beads solidify, swirl the > oPosition the comb, which is
flask again and pour melted agarose into the gel bed. aasymmetric, so its teeth are nearest
tthe black electrode.

8. Put a comb with 8 teeth in the slot at the end of the bed nearest the > Note that the gel box has three slots
negative (black) electrode. iin which a comb can be placed and
that below each slot is a red stripe
on the bottom of the gel bed. After
tthe comb has been removed, these
rred stripes will enable you to
v visualize the wells easily.

9. Allow your gel to solidify at room temperature for 20 – 30 min.. > When finished, it should look
ocludy and be firm to the touch.

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Gel Electrophoresis Pt. 2
1. Carefully remove the black dams. >
Rinse the dams well at the sink
with water from the white plastic
faucets. Put them in the slots at the
back of the gel box for storage.
2. Use the TAE (Tris Acetate EDTA) electrophoresis buffer that you
prepared by diluting the concentrated 50X buffer to make a 1X
solution.

3. Pour enough buffer into the gel chamber to completely cover your
gel. Prepare approximately 400 ml per
electrophoresis chamber and 100
ml per gel (this will give you some
4. Let the buffer sit over your gel for a few minutes while each lab
extra)
partner practices the gel loading process described next. 10 ml conc. buffer + 490 ml
distilled water
5. First, prepare the practice loading solution as described in the
sidebar to the right. To make this solution, you will use loading
dye, which contains a dye so samples are visible and a dense Practice loading solution: Pipette
180 µl H2O (clear tube) into a 1.5
reagent so samples sink to the bottoms of wells when loaded. If
ml microtube and add 20 µl loading
there are drops of dye on the sides of the tube (clear tube, purple dye (clear tube with purple dot).
dot), spin the tube for a few seconds (sec) in the microfuge before Touch the pipette tip containing the
making the practice loading solution. **some practice solutions dye to the meniscus of the water
are pre-mixed and ready to use. before expelling the dye. Close the
tube top and flick the tube
vigorously to mix.
6. Locate the practice gel.

7. Pick up 20 µl of the practice solution with your P20 micropipettor.

8. Place the pipette tip just inside the top of a well or rest the tip
against the upper edge of a well in the mock gel.
Do not actually put the tip into the
9. Push the button of the pipettor down slowly to carefully dispense well because you could puncture
the dye solution, allowing it to sink to the bottom of the well. the well and lose the sample.

10. Load one aliquot of practice loading solution, but do not load the Depressing the button of the
pipettor too quickly could cause the
rest unless you feel confident that you can do this successfully. sample to squirt out of the well.

11. When all samples are loaded, close the lid of the gel box and >
begin electrophoresis as described below.

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 12. a. Check that the power switch is off and the rheostat is turned Each power supply can run two
to the lowest setting. gels.

b. Attach the electrical leads, taking care not to jostle the box. > Connect the leads to the same
colored plugs on the power supply,
red to red and black to black. The
red lead should be at the opposite
end of the gel box from the wells of
your gel.
c. Next, turn on the power and adjust the voltage to 100 – 110 V by
turning the rheostat clockwise.

d. Small bubbles will start to rise from the electrodes if current is > If the dye does not move, or if it
flowing, and you will see the dye start to move out of the wells does not move toward the red
electrode at the far end of your gel,
within a minute or two. immediately turn the rheostat down
and the power off.
13. Continue the electrophoresis for 30 – 50 min or until the dark
blue dye has traveled to a point midway between the second and
third red stripes, about 2.5 cm from the end of the gel.

14. Turn the rheostat counterclockwise to the lowest setting, turn off
the power and unplug the leads from the supply.

>

III. Staining and Photographing Your Gel: When In our hands, Methylene Blue Plus
and FastBast are about half as
electrophoresis of your gel ends, proceed to stain your gel with sensitive as ethidium bromide but
Methylene Blue Plus as described next. has the great advantage of not
being mutagenic so you can use it
To visualize the DNA in your gel, you will stain the gel in a in your classrooms.
methylene blue based stain, Fast Blast (BioRad), or use SYBR stain
SYBR stain is a great alternative,
(pre-measured into the gel when preparing the gel – 1ul SYBR stain having almost the sensitivity f
per 10 ul of gel) ethidium bromide combined with
the safety of methylene blue based
stains.

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 > Put on gloves before proceeding.

1. Before you stain your gel, each lab pair will be assigned a number. > To avoid confusion, please use the
staining and destaining trays with
the number you have been
assigned for your gels during the
Workshop.
2.Lift the gel chamber out of the electrophoresis box and carry the
chamber to the nearest sink.

3. Use your fingers to hold your gel against the gel bed and carefully
pour the electrophoresis buffer into the sink.

5. Pour enough Methylene Blue Plus solution into a yellow staining

tray to fill it half full.
6. Transfer your gel from the gel bed to the tray by lifting the red- > Rock the tray carefully a few
striped gel bed out of the chamber and slowly sliding the gel from times to ensure the gel is
submerged in the stain.
the bed into the stain.

7. Note the number of the tray you are using.

8. In 30 min, when staining is complete, fill a white/clear destaining > As Edvotek recommends, staining
tray about half full of deionized water from the white faucets at for 30 min seems to work best.
Once the gel is well stained, DNA
the sinks. bands become visible after only a
short 15 - 20 min destaining
9. Use a spatula to remove your gel from the stain and transfer it to period. If staining is decreased to
your destaining tray containing the water. less than 30 min, a longer period
of destaining is necessary for
bands to become visible.
10. Take your gel in the destain tray to the sink.

11. Hold the gel in the tray with your fingers.

12. Pour off the destain water and rinse the gel several times with > Rinsing several times removes
fresh deionized water. excess stain.

13. Return the tray to the hood and put a twisted paper towel in the > The towel absorbs stain and
tray around the gel (not on top of it). speeds up the destaining process.

14. Let the gel destain for 10 - 30 min, changing the water a couple of
times until the background is lighter and the DNA bands are
readily visible.

15. While your gel is destaining, pour the stain back into the bottle in > Methylene Blue Plus can be
the hood. reused several times. DNA bands
stained with Methylene Blue Plus
will remain visible for several
days when gels are stored at 4o C.

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 16. Wearing gloves, carry your gel in the destaining tray to the light > Take two pictures of your gel, as
box. described next, so each partner has
a copy.

17. Use a spatula to lift your gel from the tray, tipping the spatula
slightly so excess water drains back into the tray.

18. Transfer your gel to the surface of the light box to observe and
meausre your bands. You can also use the ProScope camera to
capture an image of your gel and measure using Logger Pro.
26. Return your gel to the destaining tray and add some fresh water.

27. Put the tray in the refrigerator overnight inside a plastic Baggie. > Because the visibility of DNA
bands stained with Methylene
Blue Plus often improves with
prolonged destaining, you will let
your gel destain overnight tonight
and photograph it again tomorrow
morning.

http://www.edvotek.com

www.vernier.com

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