Lab - Week One: The Scientific Method

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Biology 211 - Lab Eight: Restriction Enzymes and Plasmid Mapping
Reminder: Bring your textbook and a calculator.
Pre-Lab Homework:
1. Name the plasmid you will be cutting/mapping in this lab. What TWO restriction
enzymes will be using in this lab?
2. What charge is DNA? How must gel be set up to make DNA "run" from wells?
What determines how far DNA will travel in the gel?
3. Name THREE DNA fingerprinting applications.
In-Lab Activity Overview
a. Setting Up Your Restriction Digests
b. Mapping Applications Problems
c. Loading Your Gels
d. Interpreting Your Gels: EXTRA CREDIT POTENTIAL!!!!!
Setting Up Your Restriction Digests
Today, you will be mapping a plasmid called pUC, which is found naturally in E. coli
bacteria. The restriction enzymes you will be cutting with are Ava II (from Anabaena
bacteria) and Pvu II (from Proteus bacteria). Each pair will set up ONE reaction (see
assignment table below). All materials for this lab cost a LOT of money and there is
NO EXTRA if any pair fails to follow directions, squanders/drops materials, or uses too
much. You should also note that three pairs will have to share a common DNA stock
and a common enzyme (or no enzyme) master mix.
Window Side of Room
Back Table/Back Pair: Ava II ONLY
Back Table/Front Pair: Ava II ONLY
Middle Table/Back Pair: Ava II ONLY
Middle Table/Front Pair: No Enzyme
Front Table/Back Pair: No Enzyme
Front Table/Front Pair: No Enzyme
Wall Side of Room
Back Table/Back Pair: Ava II & Pvu II
Back Table/Front Pair: Ava II & Pvu II
Middle Table/Back Pair: Ava II & Pvu II
Middle Table/Front Pair: Pvu II ONLY
Front Table/Back Pair: Pvu II ONLY
Front Table/Front Pair: Pvu II ONLY
Pair Procedures - DNA and enzymes must stay on ice at all times until mixed!!!!
1. Label your pair tube using shorthand to indicate who you are and your reaction
2. Using 5 ul pipetter, add 5 ul DNA to bottom of tube
3. Using 10 ul pipetter, add 10 ul of assigned master mix to bottom of tube
4. Flick tube to mix and spin using mini-centrifuge
5. Place tube in 37°C water bath and incubate at least 45 minutes (NO LESS!)
Mapping Applications Problems
1. You are studying DNA from 3 people below. The restriction enzyme cuts at the
vertical lines. Your Task: draw the DNA bands on the gel on your worksheet: Lane 1
Biology 211 Lab Manual, Lab Eight, page 1
= DNA markers (8000 bp, 4000 bp, and 2000 bp), Lane 2 = Person 1, Lane 3 = Person
2; Lane 4 = Person 3.
2. A crime has been committed!
Lane 1 is the victim's DNA collected
as a control after the crime. Lane 2
is DNA from several hairs found at
the crime scene. Lanes 3-8 are all
potential suspects. Who did it and
how do you know?
3. You are a genetic counselor working with a family with sickle cell anemia. The first
lanes are "controls" that show you the patterns for NORMAL (N), Carrier (C), and
Disease (D). Use this to draw a proper pedigree: A mother (1) and a father (2) have
five children: four daughters (3-6) and one son (7). Daughter 3 and her husband (9)
have one daughter (10). The son and his wife (8) have one daughter (11).
N
C
D
1
2
3
4
5
6
7
8
9
10
11
Biology 211 Lab Manual, Lab Eight, page 2
4. A long time ago, Dr. Boomer and her
research students studied novel bacteria from
hot springs in Yellowstone. In the following
real research gel, 10 hot spring bacteria DNA
samples (A-J) were cut with EcoRI, loaded,
and separated using gel electrophoresis.
Based on the observed patterns, how many
distinct species are there? Who is the same?
Who is different?
5. When you have completed ALL problems above, show your instructor your
worksheet. If things look complete, they will give you a gel key for solving your plasmid
mapping (i.e. what the gel should look like). If you choose to do the extra credit, you
will need this key – so hold onto it because it will not be available after today!
Loading Class Gels
Following digestion, add 5 ul of loading dye to your reaction tube, mix, and spin down.
Gels will be loaded in the following order. If your lab isn’t completely full – SKIP (as in:
leave empty) reactions that are lacking. You are strongly advised to record how the
gels were loaded in your class somewhere in your records.
Lane 1
Lane 2
Lane 3
No Enzyme No Enzyme No Enzyme
Lane 1
Pvu II
Lane 2
Pvu II
Lane 3
Pvu II
Lane 4
Ava II
Lane 5
Ava II
Lane 6
Ava II
Lane 4
Both A&P
Lane 5
Both A&P
Lane 6
Both A&P
Interpreting Your Gels: EXTRA CREDIT POTENTIAL!!!!!
Images of each class’ gels will be posted using your lecture instructor’s website by
FRIDAY (no earlier). To earn up to 5 pts., you must do turn in the following FIRST
THING MONDAY IN LECTURE (your lecture instructor will be grading it!)
1. A printed and fully hand-labeled image of your class’ gel; labeling must indicate
which lanes are which and a circle indicating which lane is your pair’s work. Indicate
which lanes, if any, are empty because of absence or not enough people in your lab.
2. A short answer-style interpretation, with each section clearly labeled:
a. uncut samples, do they match your predictions? Why or why not?
b. Ava II only samples, do they match your predictions? Why or why not?
c. Pvu II only samples, do they match your predictions? Why or why not?
d. Ava II/Pvu II samples, do they match your predictions? Why or why not?
Biology 211 Lab Manual, Lab Eight, page 3
Lab Eight: Restriction Enzymes Mapping - Turn In As Pairs At End of Lab
Pair Member Names:
1. 1 pt. Human Fingerprint Drawing; Lane 1 =
DNA markers (8000 bp, 4000 bp, and 2000 bp),
Lane 2 = Person 1, Lane 3 = Person 2; Lane 4
= Person 3.
2. 1 pt. CSI: Who did it?
3. 1 pt. Dr. Boomer’s Bacteria; How many
species? Who is the same? Who is different?
4. 2 pts. Sickle Cell Family Pedigree
REMEMBER to follow rules about pedigrees, and to label phenotype AND genotype!!!
5. 5 pts. DRAW a restriction site map for pUC19 based on Ava II and Pvu II analysis.
Restriction maps must show/label all restriction enzyme sites and label the distance (in
base pairs) between each restriction site.
Biology 211 Lab Manual, Lab Eight, page 4
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