Transcript Presentation - Cornell Institute for Biology Teachers

DNA
Electrophoresis
Cornell
Institute for
Biology
Teachers
What
is this?
How
much do
you
know
about it?
http://www.britannica.com/eb/art-40216/DNA-molecule
What are changes in
DNA
called?
1. Who thinks that DNA changes a lot?
2. Who thinks that DNA doesn’t change
much?
You are both correct.
Although large portions of human DNA
remain largely unchanged, other
portions of the molecule can change A
LOT.
We know that ONLY about
5% of the genome codes for
proteins that make up our
body. The remaining 95% of
the DNA has been called
‘junk DNA’ because it
doesn’t code for any
proteins.
However, we are learning
that it has regulatory
functions: some of this
regulates how genes
behave and what genes do. Still, it is this 95% that changes a lot and is
different in every person. This is the part that
we are going to be most interested in, and will
show us how unique each human is.
http://www.rps.psu.edu
What are
chromosomes?
Chromosomes are rod-shaped structures
made of DNA and proteins that become
visible in the cell nucleus when cells begin to
divide.
http://micro.magnet.fsu.edu
What are
genes?
Hereditary units consisting
of a sequence of DNA
Occupy a specific location
on a chromosome
Determine a particular
characteristic in an organism.
Genes undergo mutation
when their DNA
sequence changes.
http://publications.nigms.nih.gov
In the nucleus of
the cell in
eukaryotes.
Where is DNA
found?
In prokaryotes it is
in the cytoplasm –
no nucleus!
http:// cmweb.pvschools.net
http://content.answers.com
Does every cell
in the human
body have a
nucleus?
Red blood
No! cells
have
nucleus
in
Redablood
cells
thedon’t
early have
stagesaof
development, but
nucleus.
expel them as they
mature to make
room for the
So
how do
hemoglobin they
carry (remember:
they
the main function of
reproduce?
red
blood cells is
oxygen and carbon
dioxide
transportation, in
which hemoglobin is
involved).
www.irondisorders.org
How big is the
human DNA?
If you take one cell, and
stretch all 46
chromosomes end on end,
the resulting string of DNA
is about 2 meters long.
What can we do to
study such a long
molecule?
Restriction
Enzymes
More than 900 known
Isolated from bacteria
Recognize a particular
sequence and cut in a
specific position within that
sequence
Their names derive from
the organism from which
they were isolated
Example: EcoRI (a.k.a ‘eco R one’)
Isolated from certain strains of E. coli
Recognizes the sequence G-A-A-T-T-C
Cuts between the G and the A
DNA
fragments
Gel Electrophoresis Steps
Agarose
gel
fibers
1. Cut DNA
2. Make gel
3. Run DNA in the gel
Gel Electrophoresis
DNA
fragments
1.
Why does the DNA move?
2.
How do we know how far the DNA has
run?
3.
When do you stop running the gel?
4.
Why do differently sized fragments of DNA
move at different speeds?
Gel Electrophoresis
DNA
fragments
Agarose
gel fibers
-
-
+
+
-
+
1.
The DNA is negatively charged by nature, and it moves because it is attracted to the positive
end of the gel box.
2.
To make sure the DNA doesn’t run off the gel we use a loading (or indicator) dye. The dye is a
small molecule, much faster than any of the DNA fragments and is visible to the naked eye.
Because it runs faster than the DNA fragments, we can be sure that once it reaches the end of
the gel, the DNA fragments are still in the gel.
3.
When the loading dye reaches the end of the gel, we know the DNA is close to the edge as
well.
4.
The smaller fragments of DNA move faster than the heavier, larger fragments because it is
Gel Electrophoresis: Paternity Case #1
Mother
1
Child
1
2
3
Suspect
4
5
Are we sure this is not
the biological father, or
do we need more
testing?
Which is the most interesting subject in a paternity
case?
mother
This child inherited band #1 from:______________
This child inherited band #2 from:______________
Biological father
Gel Electrophoresis: Paternity Case #2
Mother
Is the suspect the
BIOLOGICAL
father of the baby,
or do we need
further testing?
1
Child
Suspect
1
3
2
2
4
This child inherited band #1 from:_____________
mother
This child inherited band #2 from:______________
Biological father
Divide the class in
working groups of 4
Make sure everyone can see the front!
Have you seen a
pipettor before?
Look at the top of your pipettor:
>
2
20
This is the range that your pipettor
can handle.
What is the unit?
Microliter (µl)
1
Decimal
point
5
0
>
microliters
Practice!
1. Set your pipettor to:
0
7
How much is this? 7
µl
0
2. Now push the control button and find
the first stop.
3. Keep pushing and find the second
stop.
Become familiar
with these
stops!
How to insert a pipette
tip
1. Open the pipette tip box
2. Insert the pipettor into the tip, and
push
3. Take the pipette and tip out of the
box and secure with your fingers
by pushing it backwards. Do not
touch the front end of the tip!
Pipettor use and safety guidelines
Pipettor Demo!
Teacher transfers 200 µl of green colored water
to another tube.
- Always hold tube at eye level
- Push control button to first stop and hold
- Submerge tip in the solution and let go of
control button (you are sucking up the
solution)
- Transfer to second tube: go to side wall of the
second tube with the pipette tip and push control
button to the first stop; hold for a second, then
push all the way to the second stop
- Holding down the control button, take out the
pipettor. If you don’t hold the control button
down before you take the pipettor out, you will suck
up some of the solution again
More
Practice
Pipettor Practice!
Working with a partner:
1.
Take a square of parafilm
2.
With the pipettor set at 7 ul, make a line of 5 drops
of green colored water on one side of the parafilm.
Remember to go to the first stop and then
submerge the tip in the solution!
3.
Have your partner do the exact same thing on the
opposite side of the parafilm
4.
Compare the drops. If they are all same size you
must have used the correct volume.
What could have happened if not all the
drops are the same size?
5. Wipe drops with with Kim wipes and discard
6. Push eject button and drop tip in the waste
basket
Teacher: collect
water tubes!
Divide each of the working
groups of 4 into two teams of 2
students
Make sure everyone can see the teacher!
1.
Establishing
Paternity
Have each team of 2 take 6 clean
Eppendorf’s from the baggie and
set on their tube rack
2. We are going to label them on
the top (lid), depending on which
case each team gets.
Case 3
3. Decide which team gets which
case and label away!
(*)
Case 4
Teacher hands out M, B
and S tubes, distilled
water and buffer, but
not the enzyme or
standard yet!
(*) will be supplied later. No tubes
need to be labeled as ‘standard’
(*)
Establishing
Go to page 8 of your handouts.
Paternity
To analyze your DNA samples, they need to be cut using a
restriction enzyme, in this case, HindIII.
Are all samples going to get HindIII? What does the “+”
stand for?
All tubes must have the exact same volume: 20
µl
Establishing
Page 8 in your handouts.
Paternity
All tubes must have the exact same volume: 20
µl
There are 6 columns to fill out. This is the list of ingredients.
What are we going to add first? ____How much in each
tube?___Remember, each tube should contain a maximum
of 20 µl of biologicals! Fill out the chart.
Establishing
Page 8 in your handouts.
Paternity
All tubes must have the exact same volume: 20
µl
Double check your amounts!
How do “13
µl” look like
in the
pipettor’s
1
window?
3
0
How do “12 µl”
look like in the
pipettor’s
window?
1
How do “2 µl”
look like in
the pipettor’s
window?
0
2
2
How do “5
µl” look like
in the
pipettor’s
0
window?
5
0
0
0
How does “1
µl” look like in
the pipettor’s
window?
0
1
0
Establishing
Page 8 in your handouts.
Paternity
All tubes must have the exact same volume: 20
µl
When pipetting, make sure you SEE what
you are adding to the tubes!
Put the substance on the side wall of
the Eppendorf (we will centrifuge it
later)
Choose one person to add the enzyme
to the “+” tubes ONLY!!! If no enzyme is
added the lab WILL NOT WORK!!
DO NOT FORGET TO CHANGE THE
PIPETTE TIPS TO AVOID
Establishing
Page 8 in your handouts.
Paternity
All tubes must have the exact same volume: 20
µl
GO AHEAD AND FILL UP ALL
THE TUBES, one by one,
following the teacher’s
instructions!!!!
Establishing
Page 9 in your handouts.
Paternity
Next, one team member brings the
tubes in the tube rack to the centrifuge,
closes the lid and centrifuges for 2
seconds and then…..
…..puts the tubes in the incubator. Pick a
letter (on the side of the incubator) to
identify the team’s tubes. Stick with
this letter so your tubes can be retrieved later!
After all tubes are in the incubator, ask a volunteer
to put the rest of the Eppendorf tubes (DNA
samples, HindIII, etc.) in the waste basket!
Establishing
Paternity
Page 9 in your handouts.
How long do samples need to be
incubated?
Don’t forget to keep track of the
time!!!!
37 ˚ C
At what temperature?
Why this temperature? Where do restriction
enzymes come from?
What reaction is taking place in the Eppendorf tubes right now?
DNA is being cut in smaller fragments
Would the reaction take place at room temperature, at about 20 ˚ C?
Probably yes, but more slowly
Would the reaction take place if the temperature was about 75 ˚ C?
Probably not; the enzyme would be denatured
Establishing
Paternity
Think about the enzymatic reaction taking place in the
incubator….
What would a graph illustrating this reaction
look like: A, B or C?
Percentage
of cut DNA
(A)
(B)
100%
50%
(C
)
Time
Establishing
Paternity
Think about the enzymatic reaction taking place in the
incubator….
What would a graph illustrating this reaction
look like: B
Percentage
of cut DNA
What would be the time at which the
reaction is completed?
100%
(B)
50%
30 min
Time
Establishing
Page 9 in your handouts.
Paternity
What is the next step in the
lab?
Prepare the buffer!Tables with the big flask also have a plastic
bottle with concentrated buffer.
1.Add 1900ml of distilled
water to the flask
2. Take 100 ml from the buffer
bottle and add to the big flask
with the 1900 ml of distilled
water.
3. Groups with the smaller 1000 ml
flask bring your flasks to groups with
the 2000 ml flasks. Mix back and forth
3 times and then take 1000 ml of the
solution. 2000 ml is enough for 2 gels!
NO SPILLS!
4. The rest of the
concentrated buffer in the
bottle (100 ml) can be used
for two more gel boxes in the
same fashion.
Establishing
Page 9 in your handouts.
Paternity
What is the next step in the lab?
Prepare the agarose!
Measure 1 gram of agarose in
a paper cup. Take into
account the weight of the cup
(tare or rezero)!
Transfer the agarose to the
small 250 ml flask.
Measure 100 ml of buffer
solution in the graduated
cylinder and add to the flask
with the agarose.
Bring the flasks to the
microwave oven and follow
teacher’s instructions.
Memorize the label on your
flask so you can recognize it
later!!
Let it boil for a minute or so
until the agar is totally
dissolved.
Establishing
Page 9 in your handouts.
Paternity
What is the next step in the lab?
Preparing the agarose….
After boiling, set all the flasks in a line….
…and let them cool down until the temperature reaches 75 ˚ C….
The teacher will add 2 µl of GelStar to each flask. Swirl
the solution.
Establishing
Page 9 in your handouts.
Paternity
What is the next step in the lab?
Preparing the gel boxes
1. Slide the top sideways and
find and remove the tray and
the comb
tray
comb
2. Firmly secure the gasket and
create a leak proof chamber
into which the liquid gel will be
poured.
Push
down
evenly
so
gaskets
don’t
pop out
of the
Establishing
Page 9 in your handouts.
Paternity
What is the next step in the lab?
Preparing the gel boxes
Pour the gel inside the gel box, inside the leak proof
chamber you just created.
Look at the comb. Find the side with the thicker
teeth and put this side of the comb in the gel.
Use the grooves towards the end of the gel tray, not
the ones in the center!
Wash the Erlenmeyer flask right
away, before the gel starts to
solidify inside!
Establishing
Page 10 in your handouts.
Paternity
What is the next step in the lab?
Loading the gels
Bring the Eppendorf tubes back from the incubator.
Look at the procedure, page 10. What are we still missing from the
tubes? What do we still need to add to the tubes, so that we know
when to stop the gel run?
Loading dye
How much do you add to each tube?
5 µl
How many pipette tips do you need?
6, one for each
Establishing
Page 11 in your handouts.
Paternity
What is the next step in the lab?
Loading the gels
Add the dye, 5 µl per tube, then put the pipette tips and used dye tube in
the waste basket.
Bring all tubes to the centrifuge and spin for 5 seconds.
Bring all tubes back to your team.
While the tubes are in the centrifuge
have a volunteer empty the waste
baskets into the trash.
Establishing
Page 10-11 in your handouts.
Paternity
What is the next step in the lab?
Loading the gels
Look at the list of samples. Which
of these are we missing?
Establishing
Page 11 in your handouts.
Paternity
What is the next step in the lab?
Loading the gels
How many wells are you going to need?
14
How many wells do you have in the gel?
20
So, be careful with the loading,
or you might run out of wells.
And if you make a mistake,
make sure to record it in your
lab handout!!
Establishing
Page 11 in your handouts.
Paternity
What is the next step in the lab?
Loading the gels
Visually divide the grayish gel into
thirds.
You will insert the pipette tip about one
third into the opaque gel….
buffer
level
1/3
1/3
1/3
Correct
Incorrect
Incorrect
Establishing
Page 11 in your handouts.
Paternity
Loading the gels
After placing the tip about one third into the gel (or half way into the
well), push the contents of the pipette to the first stop only and then
HOLD the yellow button in place! Don’t let go until you are actually
out of the buffer that covers the entire gel, or else you will suck the
sample back into the pipette tip!
1/3
1/3
1/3
Correct
Incorrect
Incorrect
(punctured)
???
?
Incorrect!
Also
punctured
Establishing
Page 11 in your handouts.
Paternity
Loading the gels
To steady your arm, place on top of the
counter….
1/3
1/3
1/3
Also, have a
classmate look at
the gel from the
side, to help guide
you, and tell you
when to STOP and
let go of the sample.
REMEMBER: Go in, push to first stop only, hold yellow button & pull
out!
Establishing
Page 11 in your handouts.
Paternity
Loading the
gels
Also!!! Locate the red
tape on the gel box.
Your wells should be
sitting on top of the
red tape, in order to
make them easier to
see from above.
After filling up the
wells, this is what they
would look like from
above.
Establishing
Page 12 in your handouts.
Paternity
Loading the gels
This is the default order in which the
samples should be loaded on to the
gel.
Go to page 12 in your handout. Use it
to take notes of what you are actually
doing.
You might have to deviate from this
order if you make a mistake. That’s OK!
Just make sure that you note what
happened next to the corresponding
well (on page 12).
If you mess up, remember: you have an extra 6
wells in the gel. And you have 25 µl of each
sample. Since you need to load only 10 µl of each
Establishing
Paternity
Loading the gels - Let’s get
Team
#3 pipettes first!
organized!
One member will be the guide, looking from the side, guiding the
student loading the sample. Switch roles after loading 3 or 4
samples, so the pipetting student now guides and the guide now
loads.
Meanwhile, Team #4 becomes the ‘helper’ team. One member
writes down the order in which the samples are being loaded on
Page 12. The other member pre-loads the pipettor: make sure that
they have the right volume setting, get a new pipette tip for each
sample and hand the loaded pipettor (with the correct sample in it)
to the student loading in the right order!
Set all pipettors to the correct volume: 10 µl
(looks like this in the window):
Decimal point
1
0
0
Establishing
Paternity
Loading the gels - Are the gels
ready?
How would you describe the gel?
When the gel is OPAQUE, it means its
ready!
Getting the gel box
ready
Getting the tray out
of the gel box:
One student holds the gel box down, another student takes out the tray
without touching the gel.
If there is a team member with nothing to do: organize the samples in the
right order in the tube rack, according to the order on page 11.
Establishing
Page 10-12 in your handouts.
Paternity
Getting the gel boxes
Find the red tape on the gel
ready
box.
Place the gel tray so that the
comb gets to sit over the red
tape. The wells are easier to
see when placed over the red
tape.
Get the buffer (you should have
about 900 ml in the flask). Fill
the side chambers of the gel
box with the buffer.
There is a black fill line on one
side. Fill to the black line.
** Make sure the red tape is
closer to the black wire!! **
Establishing
Page 10-12 in your handouts.
Paternity
Loading the
gels
Very important: Once you start loading the gels you can’t move
the gel box. Find a comfortable place where you can load the
gel; find a good angle for the loading student and for the guide.
Carefully pull the comb out of
the gel. If the gel sticks to the
comb, use the blunt end of a
pipette tip to push it down
gently. Set the comb aside.
Take turns so
everybody
loads some
samples!!
Begin loading the samples,
10 µl in each well, in the
correct order. Make sure to
note any changes to the
loading order on your lab
handout (page 12).
Establishing
Page 10-12 in your handouts.
Paternity
Loading the
When you are done loading:
gels
-every student needs to copy the notes from page 12
-put all tubes in the waste basket, then empty the waste
basket in the trash bin.
-rinse out all the glassware
Carefully put the lid back on the gel box. DON’T
RATTLE IT. Untie the cords and push the plugs
all the way into the power supply box .
Make sure black goes to black and red goes to
red. Turn on the power supply, and set the
voltage to between 185 V and 195 V.
Samples will run for about 80-90 minutes, until
the loading dye is close to the opposite end of
the gel.
Establishing
Paternity
While the gels
run…
Why is the box foggy?
Water electrolysis is taking place,
breaking the water molecules into
separate molecules of hydrogen and
oxygen which are released from the
buffer and fog up the box and the lid.
Establishing
Allele
Paternity
Allele
frequency is the relative abundance of Frequencies
an allele at a genetic place
(locus) in a population. It is expressed as a proportion or a percentage.
Victim’s blood
Victim’s blood
Evidence sample
Evidence sample
Suspect’s blood
Suspect’s blood
Frequency in
population
Chance of having these two
alleles is:
0.10
2 x 0.1 x 0.02 = 0.004
0.02
4 out of 1000 people in this
population will have this
pattern.
Frequency in
population
0.05
In NYC, for example, with a population of
10,000,000 this means 40,000 will have this
pattern
Chance of having these two
alleles is:
2 x 0.05 x 0.01 = 0.001
0.01
1 out of 1000 people in this
population will have this
pattern.
Establishing
Allele
Paternity
Frequencies
Remember:
allele frequency is the relative abundance
of an allele at a genetic
place (locus) in a population. It is expressed as a proportion or a percentage.
Victim’s blood
Evidence sample
Suspect’s blood
Frequency in
population
0.06
Chance of having these two
alleles is:
0.2
2 x 0.06 x 0.2 = 0.024
24 out of 1000 people in this
population will have this
pattern.
The chance of having all six alleles from the three loci:
0.004 x 0.001 x 0.024 = 0.000000096 = 9.6 x 10-8 ≈ 1 x 10-7
Or about 1 in ten million people in this population
will have this pattern.
In the USA, for example, with a population of
300,000,000 it means that 30 persons will
have this pattern.
Establishing Paternity (for teacher
reference)
Have a volunteer write on the board the order in
which the samples were loaded:
M3- B3- S3- M3+ B3+ S3+
#2. Have a volunteer
draw two bands for
the mother. Should
be much further
down because they
were cut (in red, just
for clarification
purposes)
#3. Have a volunteer
draw two bands for
the baby: one should
match with the
mother (in green,
just to clarify)
STANDARD
M4- B4- S4- M4+ B4+ S4+
STANDARD
#1. What does the + mean? Enzyme added
What does the - mean? Doesn’t have enzyme
What difference in size is there between these two types of samples? Which
are smaller? (The “+”. The “ – “ are bigger). Which would you find towards
the top of the gel? The “ – “, because they were not cut.
Have a volunteer draw where you would expect the bands for the first 3
samples (in yellow, just to clarify), toward the top of the gel, somewhere
similar.
#4. Have a volunteer
draw lines for the
suspect, assuming the
suspect is not the
father (in purple, just to
clarify)
#5. How many
bands are we
going to have for
the standard (see
page 6, there are
7 bands). This is
the whole
genome of the
bacteriophage
Lambda, cut with
HindIII, and the
sizes of these
fragments are
well known.
#6. Ask another
volunteer to draw
lines for M4-, B4and S4-. This is
uncut DNA,
should be along
the top of the gel,
somewhere.
#7. Repeat #2 and #3, and #4 assuming one of
the suspect’s bands matches one of the baby’s.
Establishing
Paternity
Ask: which of the bands are the most important? The baby’s because
we are trying to find something out about the baby: B3+ and B4+.
Ask: why do we run “ – “ if they are not important? They are the controls
for the action of HindIII. What would it mean if all the bands, for all M, B
and S are towards the top of the gel after electrophoresis? That they
were not cut by the enzyme.
M3- B3- S3- M3+ B3+ S3+
STANDARD
M4- B4- S4- M4+ B4+ S4+
STANDARD
x
Does this mean
the suspect is
the father? No,
just that he
cannot be ruled
out, you need
more testing.
Establishing
Go to Page 13 in your lab handout
Paternity
The DNA standard
provides DNA fragments
of known sizes that can be
used as a reference when
trying to find the size of
the unknown DNA
fragments run on the gel.
We know the size of the standard’s bands, based
on the information we get from the manufacturer
of the standard. The size of each standard band
is directly related to the distance traveled from the
well. We can now measure the distance for each
of the seven standard bands.
Establishing
Go to Page 13 in your lab handout
Paternity
What you can do now is plot the size and
distance traveled for each standard band.
x
Band size (
(23 kb)
Standard curve
kb)
x
(9 kb)
x
(6.6 kb)
x
(4.3 kb)
x
(2.3
x kb)
(2 kb)
x
(0.6 kb)
Distance traveled
(mm)
Establishing
Paternity
Go to Page 13 in your lab handout
When trying to find the approximate size of an unknown DNA fragment,
measure the distance that the fragment traveled in the gel (from the
well to where the band ended up in the gel) and use the standard curve
to determine the size of the fragment.
x
(23 kb)
Band size (
kb)
x
(9 kb)
Size ( kb)
x
(6.6 kb)
x
(4.3 kb)
x
(2.3
x kb)
(2 kb)
x
x
(0.6 kb)
Distance traveled
(mm)
Establishing
Paternity
Getting the gels out
Any allergies to
latex?
Turn everything off. Using gloves, one person will slide the
gel off the tray and put on the blue surface of the dark reader,
where it will be analyzed. Put the orange lid on top of the gel
and turn the dark reader on. Make sure the room is
darkened.
Establishing
You Paternity
should have 14 lanes to
Analyzing the gels, Case 3.
look at.
The brighter lanes (#7 and #14)
represent your……..
Standard (1)
What do M3-, B3- and S3have in common with M4-,
B4- and S4-?
They were uncut,
they are further up on
the gel.
Find the M3+ and the B3+. Do
they share a band?
Find the B3+ and the S3+. Do
they share a band? What
does this mean?
One band matches. The
suspect can’t be ruled
out. Further testing is
required to find
M3B3- S3M4- S3M3+ S3+
B4B3+
1
1
Establishing
Paternity
We are
now looking at Case 4.
Analyzing the gels, Case 4.
M3-
Find the M4+ and the B4+. Do
they share a band?
Yes
Look at the baby’s second
band.
This second band did not
come from the mother. It
came from the biological
father. Does the suspect
have this band?
No
What does it mean?
There is no match in the
suspect’s bands with the
second band of the baby
(which must have come from
the biological father).
This suspect is off the hook!
B3- S3M3+ S3+
M4+
B4+
S4+
B3+
1
1