Vampire Murder Lab
Where’s Kristen Stewart?
Disclaimer
• The third (of three) lessons in our sixth unit is devoted
to DNA technology, including the electrophoresis
process in this lab.
• However, timing of this lab means we need to start it
before we get to the lesson.
• To avoid repetition, I’ll give you the basics of
electrophoresis here, and we’ll cover it again from a
slightly different perspective in that third lesson.
• For lab report purposes, while we will do the
procedure now, the report won’t be due till after the
in-class lesson.
• Use this PowerPoint as reference.
Electrophoresis Basics
• Look at anyone else in this classroom right
now.
– Your DNA is over 99% the same.
• However, as you know, there are still some
differences.
• Those differences can allow us to compare
two samples of DNA and determine whether
they are from the same individual or not.
Electrophoresis Basics
• The problem: How do we “highlight” those
differences?
• The answer lies in restriction enzymes.
• Restriction enzymes slice DNA molecules into pieces
by making cuts at specific base sequences.
– Restriction enzymes are types of nuclease enzymes.
• Once the DNA molecule has been chopped up, there
will be fragments of DNA of varying lengths.
– If two DNA samples have the same size fragments, they
are probably from the same person. Get it?
Restriction Enzyme Example
• Here’s a sample of unknown DNA from a crime
scene, cut with restriction enzymes:
10 kb
11 kb
28 kb
4 kb
• Suppose police bring in a suspect, take a DNA
sample, and cut it up with the same enzyme:
13 kb
27 kb
13 kb
• Clearly these are not the same DNA sequences.
Restriction Enzyme Example
• Obviously, if two samples of DNA are the
same, the same restriction enzyme will
produce the same size fragments.
• FYI, the “kb” unit from the last slide is
kilobases – shorthand for units of 1000 base
pairs.
– So 14 kb is 14,000 kilobases, for example.
Electrophoresis Basics
• Here’s the thing…just because we cut the DNA
up doesn’t mean we’ll be able to actually see
how big each fragment is.
• We’ll need to separate the fragments, and
that’s where another useful component of the
DNA structure comes in handy:
– DNA is negatively charged.
Electrophoresis Basics
• Because DNA is negatively charged, it will
move in the presence of an electric current.
– In other words, if you pass a current through a
sample of DNA, it will move toward the positive
side.
– Here’s where electrophoresis comes in.
Electrophoresis Basics
• In gel electrophoresis, a sample of DNA that
has been cut with restriction enzymes is
loaded into a well (hole) in an agarose gel
rectangle.
• A current is passed through it.
• DNA fragments move toward the positive
terminal (anode).
– Smaller pieces move quickly and longer distances.
– Larger pieces move slowly and shorter distances.
• Here’s an example…
Gel Electrophoresis: Uses
• Suppose the leftmost
well in the gel to the
left is loaded with a
DNA sample from a
crime scene.
• The others are loaded
with DNA samples
from criminals.
• Whodunit?
– “I always knew Blanch
couldn’t be trusted.”
Preparation
• As you might guess, a lot of preparation goes
into the process:
– Digestion (cutting with restriction enzymes)
requires incubation.
– Dye needs to be added to the gel to see how far
the DNA fragments move.
– DNA needs to be stained to be visible after it has
moved through the gel.
– A standard should also be used to allow
measurement of DNA fragment sizes.
Standard
• Since DNA tested in electrophoresis is, by nature,
unknown, we have no reference point to judge
how big the fragments are.
• For this reason, a standard is usually added.
• It’s a sample of DNA whose fragment sizes are
known.
• We can use mathematical calculations to
determine the size of our fragments by
comparing them to the size of the standard
fragments.
– More on calculations later.
Now then…your case.
• In 2001, a 90-year-old Welsh woman was
stabbed 22 times and dismembered by an
individual that drank blood from her heart.
– This is a real case. Cue the newspaper article.
• DNA was plentiful at the scene.
• It’s your job to use the DNA from the crime
scene and compare it to the DNA of several
suspects to determine which one is the likely
culprit.
The Procedure
•
•
•
•
•
Each lab table has a class copy of the procedure.
Be sure to read ahead before performing each step.
You can reference a copy of this procedure online.
Don’t be shy – ask me if you need help.
Lastly, remember that the due date will be adjusted
as the remainder of the unit falls into place.
The Procedure: Overall
• This lab is divided into three days and thus three
large-scale segments:
• Day 1
– Casting gels.
– DNA digestion.
Divide and conquer!
Assign these jobs to your group members.
• Day 2
– Loading gels.
– Running electrophoresis.
• Day 3
– Reading/analyzing results.
The Procedure: Day 1
• Cast a gel: [Mr. G – show this stuff]
– Using masking tape and an index card, seal both
ends of a gel tray.
• The index cards are known as “dams.”
• Cut the index card down to size as closely as possible –
folding it will lead to leaks.
– Call Mr. G over. He will use a pipet to “caulk” the
sides of the tray using a thin layer of gel so
nothing leaks out when you fill it.
The Procedure: Day 1
• Pour liquid agarose gel into the tray until it’s
half-full (or half-empty if you’re a pessimist).
– Got a leak? Stop pouring and wait for the gel
you’ve already poured to solidify.
– Resume pouring. It seals itself.
• Quickly put the comb into the tray at the
position closest to the dam (either side)
before the gel solidifies.
• Give the gel between 10 and 20 minutes to
solidify.
The Procedure: Day 1
• When the gel has turned a little cloudy or
opaque, it’s ready.
• Bring the entire assembly (called a cassette) to
me.
– I’ll store it overnight.
The Procedure: Day 1
• Perform an enzyme digestion:
– The restriction enzyme is in a tube labeled ENZ
and is kept on ice.
– Which enzymes are in it?
• A mix of EcoRI and PstI.
• Say, “Eco R-one” and “PST-one.”
• We’ll talk more about these in class.
The Procedure: Day 1
• There are also some microtubes that are
color-coded:
– Green Tube: Crime scene sample of DNA.
– Blue Tube: Suspect 1
– Orange Tube: Suspect 2
– Violet Tube: Suspect 3
– Pink Tube: Suspect 4
– Yellow Tube: Suspect 5
• They already contain DNA samples.
The Procedure: Day 1
• First, you’ll add the enzyme mix
in 10 μL aliquots to the bottom
of each of the tubes.
– Use a micropipette.
– These are not disposable pipets,
but they use disposable tips to
transfer very small amounts of
liquid.
– IMPORTANT: Use a different tip for
each transfer to prevent
contamination.
• Demonstrate it, Mr. G.
– IMPORTANT: Keep the box of tips
closed when not in use.
http://www.monobind.com/site/images/Newsletter/Spring%202011%20MB%20Company%20Spotlight-web.htm?height=600&
Pause: Reading a Micropipette
• You will be using micropipettes that are adjustable
(but turning the dial) between 2 and 20 μL.
– There are 1000 μL (microliters) in 1 mL.
• Here’s how to read the dial:
2
0
4
= 20.4 μL
0
0
7
= 0.7 μL
0
8
6
= 8.6 μL
• Eject the disposable tip with the “eject” button.
Pause: Using a Micropipette
• Push the pipette onto a sterile tip.
– Don’t touch the tips with your hands.
• Push down the plunger before putting the tip
into the sample.
– Notice you can push the plunger down to two
“stop positions.”
– The first stop position is easy to reach.
– To get to the second stop position, you need to
push a bit harder on the plunger.
– Use the first stop position to get your sample.
Pause: Using a Micropipette
• Let go of the plunger.
– Watch as the sample is aspirated (drawn) into the
tip.
– Got a bubble? It’s a bad draw.
• In this case, push the plunger back down and try again.
• Move the pipette over your target.
– Push the plunger down to the second stop
position.
• That’s pipette-speak for “all the way down.”
The Procedure: Day 1
• Shake the samples or use a vortex to mix.
• Put the tubes in a mini centrifuge and run it
for a few seconds.
– The centrifuge only has an on-off switch. No
speeds.
• Turn the centrifuge off and wait for it to stop
spinning.
• Then (and only then), open the lid and take
out your sample.
Back to
The Procedure: Day 1
• So now you have a mix of DNA and enzymes
that cut up DNA, all in the bottom of the tube.
– We needed to centrifuge it to ensure that the
enzymes came in contact with the DNA.
• We want the enzymes to work well. What
should we do to increase enzyme activity?
– Heat it…but only gently!
The Procedure: Day 1
• Label a foam test tube holder with your group’s names.
• Float the tubes (in the test tube holder) in the water
bath, which is set to 37 °C.
– At this time, the enzymes are digesting the DNA and cutting
it into fragments.
• At the end of class, move the tubes to the refrigerator
in the prep room.
• Day 1 is now done. Spend some time updating your
notebooks.
The Procedure: Day 2
0
5
0
• Your digested DNA samples have been on “pause”
since last class in the fridge.
• Start the process up again by running them in the
centrifuge to bring all the liquid in the tubes down to
the bottom.
• So that you can see the samples as you place them
into the wells, add 5 μL of loading dye to each tube.
– Loading dye is labeled “LD.”
– It’s dye to help in loading the samples, hence the name.
• Shake or vortex, then run in the centrifuge.
• Your samples are now ready.
The Procedure: Day 2
• Carefully pull the comb out of the gel.
• Carefully remove the dams from the sides of
the tray.
• This needs to be done SLOWLY.
• In case you missed that line, I said, “This
needs to be done SLOWLY.”
• SLOWLY!
The Procedure: Day 2
• Carefully lay your gel in the electrophoresis
chamber.
– IMPORTANT: Red is positive; black is negative.
Lay your gel in such a way that the gel wells are
closest to the black terminal.
– Remember, the DNA will move AWAY from black.
• Get the DNA size marker (labeled M) from the
ice box.
Wait, what? DNA size marker?
• In order to analyze an electrophoresis gel and
determine the size of the DNA fragments, you
need to run a known sample of DNA through
the gel.
• This is going to be our standard.
• We can compare the size of our fragments to
the size of the standard and get a rough
estimate of how many bases long they are.
The Procedure: Day 2
• Now that your DNA has been digested by the
restriction enzyme mix and dyed, it’s time to load
it into the gel.
• As with the preparation, use a separate pipet tip
for each.
• Steady your arm, then carefully load Lane 1 with
10 μL of DNA size marker.
– The pipet tip should extend no more than halfway into
the well.
– Use a separate tip for each sample.
– Demonstrate it, Mr. G.
1
0
0
The Procedure: Day 2
2
0
0
• Do the same for the other 6 DNA samples (crime
scene and suspects), except use 20 μL.
• Remember, keep your arm steady.
• Once loading is done, fill the electrophoresis
chamber with 0.25x TAE buffer.
– This buffer carries the electric current between the
terminals.
– You should fill the chamber to just above the gel.
– Don’t pour the buffer directly onto the gel.
• Use a funnel to pour into a graduated cylinder, then pour into the
chamber.
The Procedure: Day 2
• Close the chamber lid – it only closes one way.
• Plug the cable…
• …into the power source.
– There are power sources at every other lab table,
so you may need to move.
– Each power source can accommodate up to four
chambers.
The Procedure: Day 2
• How do you set the voltage?
– You want it to be at 150 V. This one is at 300 V.
– Push the “minus” button to lower the voltage.
The Procedure: Day 2
• How do you turn it on when you’re ready?
– Make sure it’s plugged in. Then, push “Run.”
– Need to stop? Push the “Stop” button.
The Procedure: Day 2
• How do you know when you’re done?
– When a visible band of loading dye has reached
just over halfway through the gel, TURN THE
MACHINE OFF.
• You can stop it temporarily if you need to turn off one
gel but keep others running.
– Unplug chambers that are done running.
The Procedure: Day 2
• Remove the gel tray and carefully slide the gel out
of the tray and into a staining tray.
• Add 120 mL of DNA stain (called Fast Blast) to the
tray.
– The stain will help us visualize and measure the
results, since DNA is naturally colorless.
– The dye is positively charged and will bind to the
negative DNA.
– Note that the stain will also stain you. Be careful.
The List of Shameful “Do Nots”
• DO NOT orient the gel in the electrophoresis chamber
backwards.
– The DNA should start on the negative (black) side of the
chamber, otherwise it will be pulled backwards on the gel
and you get poopy results.
• You will also receive the ire of me.
• DO NOT tear your gel when removing the comb or
dams.
– Your wells will not be intact, causing the DNA to mix.
• DO NOT tear the well with the pipet tip.
– If you do, it will cause the DNA to go under the gel and ruin
the results for that lane.
The Procedure: Day 2
• Day 2 is now done. Spend some time updating
your notebooks.
The Procedure: Day 3
• Pour off the staining liquid (if necessary) into a bottle.
– This stuff can be reused, so don’t dispose of it.
• Destain your gel by washing it several times in warm
tap water with agitation in between.
–
–
–
–
Pour in warm water.
Swirl.
Pour out water.
Repeat.
• As you wash, the gel will become lighter (but never
clear), and over time the bands will become sharper.
– Even after you’re done washing.
The Procedure: Day 3
• Perform the Quantitative Analysis as
described in the lab instructions sheet.
• For this you’ll need semi-logarithmic (semilog) graph paper.
• Why semi-log paper?
– DNA moves in exponential proportion to its size.
• How is semi-log paper different? It’s all in the
increments.
– Let’s take a look at both graphing and your gels.
Reading Gels
Direction of
DNA
Movement
Ideal Gel
http://www.mun.ca/biology/scarr/gel_electrophoresis.gif
Reading Gels
Your Gel?
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Identifying Bands
• Let’s take a look at that last electrophoresis
gel as an example.
• How many bands do you see in this lane?
Identifying Bands
• I could reasonably make a case for five.
• Here’s how:
Band
Band
Band
Band
Band
Double Bands?
• Suppose you get something like this:
• When you get an extra-thick band,
thicker than your others, it’s probably
actually two bands close together.
– Measure both.
Double Band
• And speaking of measurements…
How to Measure
• Measure from the well to the band.
– Use millimeters!
“Normal” vs.
Semi-Log Paper
10000
Normal
10
9
8
7
4
3
900 1000
800
700
600
500
400
2
300
6
5
1
200
0
The X-axis is unchanged.
100
Semi-Log
How to Graph
• The distance traveled is a function of the size
of the fragments.
• Normally that would mean putting the
fragment size on the X-axis, but this time
we’re going to flip it.
– Plot “Distance in mm” across the bottom.
– Plot “Size in Base Pairs” across the left side.
• Be careful to use proper logarithmic units.
Size (base pairs)
10000
1000
100
0
5
10
Distance Traveled (mm)
15
20
About the Standard
• The DNA you loaded into the “DNA Size Marker
(M)” lane is known DNA.
– “Known” meaning we know exactly how many
fragments there will be, and how big they are.
• From your instructions, our sample will break into
six fragments of the following sizes:
–
–
–
–
–
–
23,130 bp (base pairs) – migrates shortest distance
9,416 bp
6,567 bp
4,361 bp
2,322 bp
2,027 bp – migrates longest distance
About the Standard
• Let’s add a lane
to our gel: the
standard.
• Blanch is still
the criminal,
but now we can
approximate
fragment sizes.
About the Standard
• Since we know the
fragment sizes,
let’s label them*.
• Now let’s use the
standard (or the
marker, whatever)
to estimate
Blanch’s DNA
fragment sizes.
23,130 bp
9,416 bp
~9500 bp
6,567 bp
4,361 bp
~6600 bp
2,322 bp
~2200 bp
~2100 bp
~1900 bp
2,027 bp
*not to scale
Plotting the Standard
• But it gets better: We can mathematically estimate
the fragment sizes.
• Since we know the size of the fragments in the
standard…
– And by the way, a standard is sometimes called a ladder.
• …we can plot the distances those fragments
traveled on the graph and make a curve with them.
– The curve should look “vaguely exponential.”
– I’ll plot a sample one on the next slide.
Size (base pairs)
10000
1000
100
0
5
10
Distance Traveled (mm)
15
20
Your Data
• Suppose your smallest fragment traveled a
total of 20 mm. How many base pairs in
length is the fragment?
• To approximate its length, find 20 mm across
the X-axis, then find the length that
corresponds with that point on the standard
curve.
– Here’s what I mean…
10000
1500
Size (base pairs)
1000
100
0
5
10
Distance Traveled (mm)
15
20
Conclusions
• So the DNA fragment in question is about 1500
base pairs in length.
• You can use this method to compare the crime
scene DNA with that of the suspects to find a
match and quantitatively describe the fragment
sizes.
• For your notebook:
– Identify the fragment sizes for only the
perpetrator/crime scene using the size marker lane.
Where to get the data?
• While you’ll do your own measuring (remember,
millimeters), a sample data table is in the lab
instructions found as a class copy on your desk or
available online.
• That’s where you can get the data points for the
standard.
• Note: If you really don’t see six bands for the size
marker, it’s usually better to start with band 2.
– Confused? Ask Mr. G.
The Lab Notebook
• Polish this lab off by completing it as an informal lab
report in your notebooks.
• The usual guidelines apply, but the lab instructions
document includes a series of questions that should
be answered as well.
• Note that you will also be explaining the logic
behind each step you include in the procedure
section of your report.
• Don’t worry about getting the right suspect.
– There are no points associated with being right/wrong.
– Just explain yourself and you’ll be fine.