Electrophoresis LAB/Chapter 11 Packet
Name ___________________
Date ___________ Period___
Electrophoresis Quiz Study Guide
1. Where is DNA found?
2. What barriers need to broken to get to DNA?
3. What techniques were used in the DNA Virtual Extraction Lab to break through the barriers?
4. What type of cells would you find DNA in at a crime scene?
5. What technique do scientists use to make more copies of DNA from a single cell?
6. What are restriction enzymes?
7. Why do restriction enzymes cut different peoples DNA at different places?
8. What is DNA fingerprinting?
9. How are restriction enzymes used in DNA fingerprinting?
10. What are some uses for DNA fingerprinting?
11. What is electrophoresis?
12. How does electrophoresis help with DNA fingerprinting?
13. What are 5 sources of error from DNA electrophoresis?
14. How are restriction enzymes used to insert human DNA into another gene?
15. What are exact copies of organisms called?
Vocabulary
Restriction enzymes
Electrophoresis
Anode
Agarose
Comb
Wells
Cathode
Glycerol
Orange G
Buffer
DNA Ladder
Methylene Blue
Light box
DNA Extraction Virtual Lab (Web Activity)
Follow the directions on the website. Make sure to watch everything that goes on as much as possible. If you miss
something, click on the back arrow on the slides in the website. Write your answers as completely as possible
below. 1. Give 3 reasons why we might extract DNA from humans. ______________________________
2. Why is important to purify DNA? ___________________________________________________
3. What organelle in the cell is DNA found in? ________________________________________________
4. How many meters of DNA is in each cell? ______________________________________
5. List the four steps that you will follow to purify DNA from cells.
1. ___________________________________________________
2. ___________________________________________________
3. ___________________________________________________
4. ___________________________________________________
6. List at least 6 of the items you will need to isolate DNA. _______________________________________
_____________________________________________________________________________________
7. What does lysis mean? ___________________________________________________________
8. Why did we add the detergent and the protienase K?
Detergent-___________________________________________________________________
Protienase K- ________________________________________________________________
9. Why do we add the concentrated salt solution? ____________________________________________
10. What do you have to do in order to balance the centrifuge? ___________________________________
11. After the tube is spun in the centrifuge, where should the DNA be? ___________________________
12. Why do you add the isopropyl solution and invert the tube? _____________________________
13. Once you remove the liquid how long can the DNA last if stored in the freezer? _________________
PCR Virtual Lab (Web Activity) Follow the directions on the website. Make sure to watch everything that
goes on as much as possible. If you miss something, click on the back arrow on the slides in the website. Write your
answers as completely as possible below.
1. What does PCR stand for? _______________________________________________________
2. What is the main use for PCR? _______________________________________________________
3. Give four uses for PCR given. _______________________________________________________
Click Begin when you are ready
4. The human genome is made up of how many base pairs? __________________________________
5. Click NEXT  The picture shoes that ______ is found in the nucleus.
6. Click NEXT You need ______ extracted from cells.
7. This can be found in ____________, ______________, ________________, or ___________ cells.
8. Click OKAY PCR tubes are special in that it allows for _______ and ______ over and over again.
9. Click OKAY Drag the DNA to the PCR tube and the click to release it.
10. Click OKAY Primers attach to the _______ of DNA. They help to copy DNA.
11. Click OKAY Drag primer 1 to the PCR tube and the click to release it.
12. Click OKAY Drag primer 2 to the PCR tube and the click to release it.
13. Click OKAY What are the four nucleotides in DNA? ______ _____ ______ _______
14. Drag the nucleotides to the PCR tube and the click to release it.
15. Click OKAY What are DNA Polymerases like? ______What do they do? ___________________
16. Click OKAY Drag DNA Polymerase to the PCR tube and the click to release it.
17. Now Primers 1 and 2, the nucleotides, and DNA Polymerase are in the tube.
18. The machine that heats and cools the DNA at specific times is called the _________ ____________.
19. Click and Drag the PCR into the machine called the _______________ __________________.
Draw a picture of the DNA here.
20. Click START In cycle one, the machine heats up to ____ C,
which equals ___ F (close to boiling).
21. This causes the DNA strands to __________ .
Click NEXT The thermal cycler will cools down to ____ C,
which equals ___ Fahrenheit.
Draw a picture of the DNA below
22. This makes the two DNA strands ______________.
But there are a lot more primers in there, than _______.
This makes the ____________insert themselves into
Add primers/DNA polymerase above.
the strands, before they rejoin.
Click NEXTChanging the temperature to 72 C activates
__________ __________________, which
adds ____________ to the ends of the primers.
23. This continues until it gets to the end, where it _________.
Draw the finished DNA strands above
24. Click NEXT- Cycle 1 is complete- Click NEXT to begin Cycle 2 The same ____ steps happen in cycle two. The
temperature is raised again to __________________________________________.
25. Click NEXTThe temperature is lowered so the ________________________________________.
26. Click NEXTThe temperature is raised again to activate _____ _________, which will make more___.
27. Click NEXT to begin Cycle 3 The desired products begin to appear at ________________.
28. There are ____ fragments now .
29. Click NEXT  At the end of cycle 4 there are ___ fragments.
30. Click NEXT How many are there after 30 cycles? __________________________________
DNA Electrophoresis Virtual Lab
(Web Activity)
Follow the directions on the website. Make sure to watch everything that goes on as much as possible. If
you miss something, click on the back arrow on the slides in the website. Write your answers as
completely as possible below.
1. What is your job? ___________________________________________________
2. What process do scientists use to sort DNA (and proteins) by length? _________________________
3. What is the filter that is spongy and has a lot of holes in it called? _________________________
4. We put the _________ samples in to the holes or wells at one end of the gel.
5. What do you have to add in order to make the DNA move? _________________________
6. Which electrode (charge) is closest to the DNA? _________________________
7. Towards which electrode (charge) will DNA move? _________________________
8. Which strands move faster? __________________________________________________
9. Why do we stain the DNA? __________________________________________________
10. List the 5 steps to run your own electrophoresis experiment (at the top of slide).
1. __________________________________________________
2. __________________________________________________
3. __________________________________________________
4. __________________________________________________
5. __________________________________________________
11. What are the 6 things you will need to make the gel? ________________________________________
_______________________________________________________________________________________
12. What is agarose? ____________________________________________________________________
13. Why do we need salt water buffer in electrophoresis? _______________________________________
14. What is the purpose of the comb? __________________________________________________
15. What are two reasons for adding the loading buffer to your DNA samples? _____________________
_______________________________________________________________________________________
16. Why do we need to have a clean pipette tip? (this is not from the website, just common sense)
___________________________________________________________________________
17. What does the DNA standard contain? __________________________________________________
18. Why is it important to run the standard with your gel? ______________________________________
_______________________________________________________________________________________
19. What color is the negative electrode?_______ What color is the positive electrode? __________
20. What charge does DNA have? __________ What color do you want closest to the wells? _________
21. What do you need to look for to show that current is moving through your gel? _________________
22. Why does DNA move towards the positive charge? ______________________________________
23. Why do we need to stain the DNA? __________________________________________________
24. What does the UV box help with? ______________________________________________________
25. Write down your estimates for the 3 pieces of DNA in the sample. _______bp , _______bp , _______bp
Chapter 11 Reading Guide
Pages 228-232: Genetic Engineering
1. What did S. Cohen and H. Boyer do in 1973 that revolutionized genetic studies in biology? ________
_________________________________________________________________________________
2. What was the first genetically altered organism (see figure 1)? ______________________
3. The process of manipulating genes for practical purposes called ___________ ______________.
4. DNA made from two or more different organisms is called _________________ _____.
5. How did in people who are diabetic get insulin before genetic engineering?
6. What is the human insulin gene transferred into through genetic engineering? ______________
7. List the steps in a Genetic Engineering Experiment. (copy titles from picture on page 229)
1. ____________________________________________
2. ____________________________________________
3. ____________________________________________
4. ____________________________________________
8. Bacterial enzymes that recognize and bind to specific short sequences of DNA, and then cut the DNA
between specific nucleotides within the sequences are called _________________ ________________.
9. A _____________ is an agent (organism/cell) that is used to carry the gene of interest.
10. Circular pieces of DNA in bacteria cells are called _____________. Theses molecules can usually make
copies of itself independently of the main ________________________ in bacteria.
11. Since both the gene of interest and the vector are cut with the same enzyme (chemical scissors), they can be
bonded together using the enzyme _________ ___________________. Then the host cell takes up the
____________________ __________.
12. Many identical copies of the gene are made in _________ __________________. Since bacteria reproduce
asexually through ____________ _________________, all of its offspring are _________________.
13. Scientists have to have a _________________ process to see which cells picked up the gene of interest and
which ones _________________.
Pg. 230
14. An example of how the ____________________ ___________ ECORI is in Figure 3.
Draw your own picture here from figure 3 to help you understand how it works.
15. What is a palindrome? ________________________________________________________________
16. What is a sticky end (use the words-- complementary bases, cut, restriction enzymes and single strands in
your definition)? _________________________________________________________________________
17. In your own words explain how restrictions enzymes allow genes to be inserted into vectors. ___________
________________________________________________________________________________________
18. Finding which cells have taken up new genes is important to scientists. What is one way they do this? ___
________________________________________________________________________________________
Page 231
19. List the four steps of a southern blot (copy titles from picture on page 231)
1. ____________________________________________
2. ____________________________________________
3. ____________________________________________
4. ____________________________________________
20. In the process called a ____________ _____, DNA from each bacterial clone colony is isolated and cut into
pieces by __________________ ____________.
21. A technique that uses an electric field within a gel to separate molecules by size is called _____________.
22. What charge does DNA have? ____________________________________________________________
23. Which DNA piece move the fastest? _______________________________________________________
24. Radioactive or fluorescent labeled DNA or RNA that is complementary to a gene of interest are called ____.
25. Only the DNA/RNA strands that are complementary to the ______________ will show up.
Pages 233-237: Human Applications of Genetic Engineering
26. What is the Human Genome project? ______________________________________________________
27. What are 2 major goals of this project? ____________________________________________________
28. Describe how drugs produced by genetic engineering are being used. ______________________________
29. What is a vaccine? _____________________________________________________________________
30. How do vaccine’s made by genetic engineering avoid the chance of giving the person the disease when they
get the vaccine? _______________________________________________________________________
Page 236 (PCR)
1. What technique do scientists use if they need to make many copies of DNA? ________________________
2. Why is the DNA heated first? _____________________________________________________________
3. After it is cooled, what is added? __________________________________________________________
4. What do these (answer to 3) things do? _____________________________________________________
5. What does the DNA polymerase do (think back to DNA replication)? _____________________________
6. The process is repeatedly heated and cooled. How long does it take for the sample of DNA to double? ___
________________________________________________________________________________________
7. List four ways PCR is important and used for.
1. _______________________________________________________________________________
2. _______________________________________________________________________________
3. _______________________________________________________________________________
4. _______________________________________________________________________________
Page 237
8. Who are the only two individuals that have the same genetic material? ____________________________
9. What is used to cut DNA? ________________________________________________________________
10. What is an RFLP? ______________________________________________________________________
11. Give 2 reasons why are RFLPs important to DNA Fingerprinting. ________________________________
12. A pattern of dark bands on photographic film that shows an individuals RFLP profile is called a ________.
13. Do restriction enzymes cut different individuals DNA into pieces of the same length? ___________
14. List four places DNA samples can be taken from. ______________________________________
15. The scientific investigation of the causes of injury and death when criminal activity is suspected is ______.
16. Reread page 237. List 4 ways DNA fingerprints are used. _______________________________________
Pages 238-242: Genetic Engineering in Agriculture
17. List 4 ways genetic engineers have been able to alter genes in plants in order to improve them in terms of
their environment? _________________________________________________________________________
18. How have genetic engineers been able to improve the nutritional value of foods? ____________________
Page 239
19. What are GM crops? ____________________________________________________________________
20. What are 2 potential problems with GM crops? _______________________________________________
21. Are GM crops harmful to the environment? (You give our opinion and support it with details from this
section after reading it).____________________________________________________________________
Page 240
22. What do farmers add to the diet of cows to increase milk production? ___________________________
23. Animals that have foreign DNA in their cells are called _________________ ________________.
24. Read pages 241-242. Summarize the cloning of sheep through the use of differentiated cells.
DNA Electrophoresis Lab Background Information
When DNA is digested (cut) by restriction enzymes, the result is a DNA solution containing DNA
fragments (pieces) of varying sizes. Restriction enzymes are proteins produced by bacteria that cut
foreign DNA into pieces. The number of fragments and the sizes of the fragments depend on the
restriction enzyme used and the size of the original DNA molecule.
The first is accomplished by separating the DNA using
agarose gel electrophoresis. Electrophoresis is the
process used to move a charged molecule (DNA) in
an electrical field. Its purpose is to separate the pieces
by size. A charged molecule will migrate toward the
electrode of opposite charge. Since DNA molecules are
negatively charged, they will migrate toward the
anode (positive electrode).
wells
Large
fragment size
In order to determine what the DNA fragment sizes are, it is necessary to:
(1) separate the fragments by size
(2) have some way to visualize the DNA
(3) have a standard to which the fragments can be
compared.
Small
Gel box
Casting
tray The gel or matrix such we will use is agarose, which
is used to separate most DNA molecules. It will act as
a sieve and separate the DNA molecules based on
their size. Agarose is a polysaccharide (from algae)
that can be dissolved in hot water (it is clear when
heated). As the agarose solution cools, it solidifies to
form a matrix of gelatin-like consistency (and turns
opaque). The matrix contains pores through which the
DNA molecules must pass. The size of the pores, and
hence the sizes of the DNA molecules that can be
separated on the gel, is the concentration of the
agarose solution.
It is necessary to have some method for visualizing the
DNA in the agarose gel. In this lab, either methylene
blue or Bio-Safe is used as a post-stain for DNA. The
dye is inserted between the stacked bases of DNA and
appears blue when the gel is exposed to UV light. The
DNA bands appear blue on a clear background and the
migration of the fragments can be measured. By
recording the distances the pieces of DNA (of known
length) in the standard have traveled, we can figure
out how big the pieces in our unknown samples are.
Using the migration distances of the DNA fragments in
the standard, a standard curve can be generated and
the sizes of the DNA fragments from the experimental
restriction digests can be calculated.
fragment size
Casting
Comb
gates
As the gels are prepared, a comb is placed in the gel at the
end closest to the cathode (negative electrode). After the
agarose solution has solidified, the comb can be removed,
leaving small holes or wells in the gel into which the samples
will be loaded. The DNA samples are mixed with a loading
buffer that contains glycerol and a tracking dye. The glycerol
adds density (weight) to the samples, assuring that they will
stay in the wells when loaded. The tracking dye usually
contains a dye like Orange G, which migrates through the gel
at a position approximately equivalent to a DNA fragment of 50 base pairs. The dyes serve two
functions. They makes it easier to see the samples while the wells are being loaded and, since the
dye can be seen as it migrates through the gel, it can be used to estimate how far the DNA has
migrated in the gel.
cathode
When it is time to load and run the gel, the gel is covered in
wells
buffer, the comb carefully removed, and the samples loaded
into the wells. The buffer that is added helps to spread the
Large
electrical charge or current evenly over the entire gel. A
standard solution consisting of DNA fragments of known
sizes is loaded into an adjacent well. This standard
DNA
solution is called the DNA ladder. It is called this becauseladder
of what it looks like when it separates on the gel. The lid is
placed on the gel box, the gel box is connected to a power
supply, and an electrical current is passed through the gel.
The DNA molecules immediately begin to migrate toward the anode,
Small
with smaller molecules migrating more rapidly than larger DNA molecules
anode
DNA DETECTIVES OR “WHO DUNNIT?”
Pat Neeley, Jefferson Forest High School, Forest, VA
Introduction: Many of the revolutionary changes that have occurred in biology over the past fifteen years can be
attributed to the ability to manipulate DNA in defined ways. The principal tools for the recombinant DNA technology are
enzymes that can “cut and paste” DNA or restriction enzymes. A sample of someone’s DNA, incubated with restriction
enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a
different nucleotide sequence and would thus be enzymatically “chopped up” into a very different collection of fragments.
Because no two people (except identical twins) have exactly the same DNA, a person’s DNA fingerprint is unique and
can be used for the purposes of identification. We have been asked to apply DNA fingerprinting to determine which
suspect should be charged with a crime perpetrated in our city.
WHO DUNNIT?
A murder has been committed, and police discover evidence of a struggle and blood traces at the scene of the
crime. Ian, a UPS delivery man, is found dead in his truck on Rt. 221 just west of Forest Middle School.
Autopsy has shown that Ian was strangled to death, but there is no blood from the victim on the scene. Packages
are missing from the truck, and no witnesses can be found. Suspects X, Y, and Z are arrested and will go
through DNA tests to determine if they were at the scene of the crime. What DNA sources other than blood
might be found at the crime scene? All of the suspects proclaim their innocence adamantly, and all want to see
their lawyers. At their indictments, it is learned that:
□ Suspect X - Bob Smith is a man in his middle thirties with prior convictions for armed robbery. Bob was
apprehended shortly after the murder in Bedford driving recklessly on an expired license. No contraband
was found in his possession but his hands are cut in several places. He says it’s because he works
construction.
□ Suspect Y - Jim Dale is a man in his late forties. He is suspected of being romantically involved with
Ian’s wife, Pam. Unexplained scratches were found on the back of his neck.
□ Suspect Z - Pam, wife of Ian. She says she was with Jim the entire day. Several cuts on both hands are
suspicious. She claims she got them while picking blackberries with Ian.
You are the lab worker who has been handed the DNA samples from the three suspects involved, plus the DNA
from the blood at the crime scene. Using molecular biology techniques, your job is to determine which of the
suspects might have been at the crime scene. The court awaits your findings.
1.
2.
3.
4.
5.
6.
7.
Place the Casting Tray in the Gel Box (make sure notches match up)
Insert the casting gates and make sure there is a good fit.
Place the comb in the tray in the slots closest to the black (cathode) end.
Swirl the gel, make sure that it is completely melted.
Pour quickly and make sure the tray is not disturbed at all.
When gel is opaque, remove the casting gates and comb.
Pour the buffer over the gel, filling both wells and
covering the gel completely.
8. Follow the guide to the right to load the wells.
9. Use a new tip for each sample
10. Put the lid on in the correct way.
11. Plug in the Lid into the power supply
12. Hit Run, look for the bubbles to make sure it is running.
13. Watch the dye and unplug it when it nears the positive end.
14. Let the teacher know that your gel is finished.
15. Place the labeled bag on the chamber.
DNA Electrophoresis Study Guide Questions
1. What is electrophoresis? _________________________________________________________
______________________________________________________________________________
2. What is its purpose? ____________________________________________________________
______________________________________________________________________________
3. What are restriction enzymes?_____________________________________________________
________________________________________________________________________________
4. Why must restriction enzymes be added to DNA?_____________________________________
________________________________________________________________________________
5. What electrical charge does DNA have? ____________________________________________
6. Towards what electrical charge will DNA move and WHY? ____________________________
7. What is the negative electrode called and what color is it? ______________________________
8. What is the positive electrode called and what color is it? _______________________________
9. What size molecules move the fastest? ______________________________________________
10. What is the name of the type of gel we are using? _____________________________________
11. What is the gel made of? ________________________________________________________
12. What color is the gel when it is cooled? _____________________________________________
13. What is added to the gel to make the wells? __________________________________________
14. What are the wells? _____________________________________________________________
15. What two things are in the loading buffer that is added to the DNA samples?________________
16. What are two reasons why it is important to add Orange G? _____________________________
____________________________________________________________________________
17. Why is glycerol important? ______________________________________________________
18. What is the DNA ladder (loaded in the end lane)? ____________________________________
____________________________________________________________________________
19. Why is it important to have the DNA ladder? ________________________________________
____________________________________________________________________________
20. What type of stain is used once the electrophoresis is complete? _________________________
21. Put the following steps of gel electrophoresis and analysis in the correct order.
Lab (1-11)
___Put the lid on the chamber and plug the correct electrodes into the power box.
___Remove stain and de-stain with small amounts of water until bands are clear.
___Load the DNA samples, changing tips between each sample.
_4_Remove the comb carefully to avoid tearing (pull straight up).
___Turn the power off when the dye front reaches close to the end.
___Pour the agarose gel quickly and let it cool until opaque without jarring.
___Put the casting tray into the chamber with the comb slots closest to the black electrode.
___Pour buffer to cover the gel completely (there should be no dimples over the gel).
_8_Turn on the power box and look for bubbles showing electricity passing through the chamber.
___Place the casting gates on both sides and the comb in the tray slots closest to the black electrodes.
___Remove gel from chamber, slide into tray and add stain for 20 minutes.
Analysis (1-4)
___Match the bp lengths of the crime scene evidence with the suspects to determine Whodunnit.
_2_Create a standard graph from DNA ladder on computer.
___Trace gel and measure distance each band has traveled from the well .
___Record base pair (bp) length
Can DNA Demand a Verdict? (Web Activity)
1. What year was DNA Analysis first scene in the courtroom? _______________________
2. What was it originally called? ______________________________________________
3. What are two names it is known by today? ______________________________________________
4. What percentage of court cases is this used in? _______________________
5. On average what percentage of DNA do most people share? ______ What percentage to identical twins share? ___
6. How many base pairs does each cell contain? ______________________ What is 0.1 % of this? ______________
7. Is this enough to identify a person? __________________________
8. How many total bases are there in the picture to the right? ______
9. How many total base pairs are there? ___________
10. DNA length is measured in base pairs. Circle a base pair in the picture.
11. Give 5 examples of DNA left behind at a crime scene. ______________________________________________
____________________________________________________________________________________________
12. What process allows scientists to make millions of copies of DNA? _____________________________________
13. How is this helpful for minute amounts found at a crime scene? _________________________________________
14. Do blood relatives share more DNA or less? ______________________________________________
15. List three ways detectives can reduce or eliminate contamination of samples. ______________________________
____________________________________________________________________________________________
16. Using the diagram on the website and the pictures below, list the steps (in your own words) that are used to collect
and analyze DNA evidence.
1.
1.
2.
2.
3.
3.
4.
4.
5.
5.
6.
6.
7.
7.
17. List 3 types of evidence other than DNA that help to link a suspect to a crime. ____________________________
18. Explain why multiple pieces of evidence pointing to the same person is good for investigators. _____________