BIO 2 Laboratory
Spring 2008
LAB PRACTICAL EXAM
(100 points)
KEY
1.
(6 pts)
You wish to make 250 mL of a 20 mM solution of this water-soluble reagent.
a.
Calculate how many grams of the reagent you will need to weigh out. Show
your entire calculation, including all units, for full credit.
(136.09 g/mol)(20 mmol/L)(1 mol/1000 mmol)(0.250 L) = 0.68 g
b.
Describe the steps you would take to make up the solution. (Assume that the
solvent is dI water.)
Add 0.68 g of the powdered reagent to 200 mL dI water in a beaker. Stir to
dissolve and then transfer the solution to a graduated cylinder that can
accommodate at least 250 mL. Top off the solution with dI water to a final
volume of 250 mL.
2.
(4 pts)
You may touch the micropipettor, but DO NOT ADJUST THE DIAL.
a.
What is the volume setting? 945 uL = 0.945 mL = 0.000945 L
b.
What is the upper limit on the volume that this micropipettor can accurately
measure?
1,000 uL
3.
(4 pts)
A plate has 130 E. coli colonies and was prepared from a culture of E. coli containing
3.4 x 105 cells/mL that had been diluted 1000-fold prior to plating. How much
volume of the diluted culture was spread on the plate? Show your entire calculation
for full credit, including all units, and express your final plating volume in uL.
Conc
(undiluted)
= (# colonies)(1000) ; Vol plated = (# colonies)1000)
vol plated
Conc (undiluted)
Vol plated = (130 colonies)(1000) = 0.382 mL = 382 uL
3.4 x 105 cells/mL
4.
(5 pts)
The kitten on the left (Copy Cat) is a clone of the cat on the right (Spice Girl).
Explain how a kitten that looks like Copy Cat could possibly be the clone of a cat that
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BIO 2 Laboratory
Spring 2008
looks like Spice Girl. Draw a diagram of how nuclear injection cloning works to help
you provide a full explanation.
Spice Girl is a tortoise shell cat. She has a gene ("O") coding for an enzyme that
turns black pigment orange on one of her X chromosomes and a gene ('o") coding
for an inactive version of this enzyme on the other. Due to X-inactivation early in the
development of female mammals, some of her cells have the "O" turned on and
express orange pigment and some have the "o" turned on and express black pigment.
(She is BB or Bb for an autosomal gene coding for black pigment, so in the absence
of "O" black fur color will result.) When cloning Spice Girl, only a single cell was
chosen. In this case, the cell used to clone Copy Cat had the "o" X chromosome
turned on. This X-inactivation was maintained through embryogenesis of Copy Cat,
so she is solid black (all of her cells express the "o" allele only).
*5. (6 pts)
You have two solutions. Solution A is 2 M Tris-EDTA and Solution B is 150 mM KCl.
How would prepare 100 mL of a mixture of these solutions such that the final
concentration of Tris-EDTA is 50 mM and the final concentration of KCl is 20 mM?
Show you calculations and write down the final recipe in "cookbook" format,
assuming that the solvent is dI water.
Vi = (Cf)(Vf) ; For Tris-EDTA: Vi = (50 mM)(100 mL) = 2.5 mL
Ci
2000 mM
For KCl: Vi = (20 mM)(100 mL) = 13.3 mL
150 mM
2.5 mL
13.3 mL
84.2 mL
100 mL
*6.
2M Tris-EDTA
150 mM KCl
dI water
(3 pts)
You extract DNA from a metallic green colony taken from an EMB agar plate.
After DNA extraction, you add approximately 50 ng of the DNA to a PCR
reaction containing primers that target a gene common to all coliforms. You then run
the PCR reaction on a 2% agarose gel.
Would you expect to see a band in the lane containing your PCR reaction? Explain.
Yes. E. coli is a coliform and therefore a DNA extract from E. coli should give a
productive PCR reaction when targeted by primers that amplify genes common to all
coliforms.
*7.
(6 pts)
You wish to determine if the bacteria from the urine of a female patient with an E.
coli urinary tract infection carries the gene for ampicillin resistance, thus making the
infection itself resistant to the drug. To begin the experiment, you make serial
dilutions of urine from the patient, plate them onto EMB agar, extract DNA from one
of the E. coli colonies, and then analyze the extract using PCR.
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BIO 2 Laboratory
a.
Spring 2008
Describe one positive control you could use for this experiment. List the
primers you would use (gene targeted in PCR) as well as the sample (DNA
extract type, water, etc.) you would test.
Amplify a DNA extract from an E. coli strain known to be ampicillin resistant
using primers targeting the gene that confers ampicillin resistance.
b.
Describe two negative controls that you would use for this experiment. In
each case, list the primers you would use (gene targeted in PCR) as well as
the sample (DNA extract type, water, etc.) you would test.
Amplify a DNA extract from an E. coli strain known to be ampicillin sensitive
using primers targeting the gene that confers ampicillin resistance.
Amplify the nuclease-free water used to set up the PCR reactions with
primers targeting the gene that confers ampicillin resistance.
8.
(4 pts)
Examine the prepared slide under the microscope. This shows something that you
observed during one of the BIO 2 laboratories. What are you looking at? How do
you know?
Cheek cells with adhering bacteria. The cheek cells are large while the bacteria are
very small (approximately 1/20th of the diameter of the cheek cells).
*9.
(4 points)
A restriction enzyme has a 10 base-pair palindromic recognition site.
a.
How many times would this enzyme cut the human genome? (The human
genome is 3.3 x 109 base-pairs.) Show your calculation.
(3.3 x 109)
410
b.
= 3,147
In the space below, draw a double-stranded 10-bp palindromic DNA sequence.
Use all 4 DNA nucleotides (A, C, T, and G) in your hypothetical sequence and
label the 5' and 3' ends of each strand of the DNA.
5' - ACTGCCGGCAGT - 3'
3' - TGACGGCCGTCA - 5'
*10.
(4 pts)
Briefly explain how ethidium bromide works as a stain for visualizing DNA.
Ethidium bromide is a fluorescent dye that intercalates between the stacked basepairs of double-stranded DNA. Ethidium bromide is excited by UV light and will
fluoresce on a UV transilluminator, allowing the researcher to visualize the DNA.
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BIO 2 Laboratory
*11.
Spring 2008
(4 pts)
Calculate the final concentrations of DNA and dNTPs in this mixture. Show how you
made your calculations.
10
5
85
100
uL
uL
uL
uL
0.2 ng/uL DNA
2 mM dNTPs
sterile, distilled water
Cf = (Ci)(Vi) ; For the DNA: Cf = (0.2 ng/uL)(10 uL) = 0.02 ng/uL
Vf
100 uL
For the dNTPs: Cf = (2 mM)(5 uL) = 0.1 mM
100 uL
*12.
(3 points)
What is the reason that RNA must be treated with urea or formamide in order to
determine the molecular weight of the RNA molecule(s) by gel electrophoresis?
RNA is single-stranded and folds back on itself to form complex secondary structures
stabilized by Watson-Crick base-pairing. Since conformation can affect the
movement of RNA molecules into the gel, this secondary structure must be
denatured in order to determine the size of RNA molecules on a gel. Urea and
formamide are both denaturing agents that break hydrogen bonds.
*13.
(2 points)
How big would a genome need to be such that the following sequence would only be
found (by random chance) one time?
5' - A T C G G G C - 3'
1 cut = 16,384 bp
47bp/cut
*14.
(6 pts)
In each case, read the hypothesis and state one thing that is wrong with it and one
thing that is good about it.
1.
Do bald eagles inhabiting the northern quadrant of Denali National Park have
significantly larger broods during years in which the total recorded rainfall in
the region is high (9-11 inches) than they do in years in which the
recorded rainfall is low (0-5 inches)?
Wrong: It's written in the form of a question rather than a statement.
Good: It provides a well-defined and testable dependent variable.
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BIO 2 Laboratory
2.
Spring 2008
There are fewer moons orbiting Mars in even years than there are in odd
years.
Wrong: It is not based on prior observations and knowledge, and is therefore
clearly absurd. Try getting funding to run this experiment!!
Good: The dependent variable is easy to assay using Earth-based telescopes.
*15.
(2 points)
The primers for a PCR reaction are listed in a Materials and Methods section of a
paper as follows:
CFTRe1f:
CFTRe1r:
5' - C C G A G A T C G A T A T A G A T A G C T A G - 3' (2876-2898)
5' - A T T C C T A T C G C G A T C C G A C A T G - 3' (3677-3656)
Calculate the size of the PCR product that would be amplified by this primer set.
Show your calculation.
3677 - 2876 + 1 = 802 bp
*16.
(3 points)
The crotch area of a sexual assault victim's panties was screened for the presence of
semen.
a.
Describe how you would perform the test. Be sure to include the name(s) of
the reagent(s) you would use.
Saturate a piece of filter paper with distilled water and then place it over the
crotch area of the panties with gentle pressure with a gloved hand. Then
spray the piece of filter paper with AP spot solution.
Alternatively, you could saturate a sterile cotton swab with distilled water, rub
it across the crotch area of the panties, and then saturate the swab with AP
spot solution.
In either case, the white filter paper or swab surface will turn pale purple if
semen is present.
b.
17.
What specific enzyme was being screened for in the assay? acid phophatase
(4 pts)
This is a photograph of a very abnormal infant who only lived for a few hours.
a.
How many chromosomes did this baby have in each of its cells? 69
b.
What event most likely caused the baby to have this unusual karyotype?
Dispermic fertilization. (Two sperm entered the same egg at fertilization.)
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BIO 2 Laboratory
*18.
Spring 2008
(4 pts)
Diana has recently been diagnosed with uterine cancer at the young age of 22. She
knows that if she survives the disease she definitely wants to have children some
day but that her eggs will be destroyed by the chemotherapy and her uterus is going
to have to be surgically removed. What could she do now to ensure that she will be
able to have her own genetic children at some point in the future? What will she
need to do in the future to make it happen?
Diana's eggs are still young, so she could take hormones that would cause her to
superovulate, and her eggs could then be removed from her ovaries and frozen until
she is ready to have a baby.
Since she has no uterus, she will have to find a surrogate to carry her child. Her egg
would have to be unfrozen and fertilized in a petri dish (presumably using her
partner's sperm) and then transferred to the surrogate for gestation and birth.
19.
(2 pts)
For what purpose did we use these reagents in lab?
To stain the cellular extract from a vaginal swab to see if it contained spermatozoa.
*20.
(4 pts)
April and Anthony are both carriers for a fatal childhood autosomal recessive disease
called Tay Sachs Disease. They would like to have children who are genetically theirs
but do not want to have a child with Tay Sachs. What could this couple do to ensure
hat they will have a healthy baby as well as one who is not a carrier for the disease
allele? In your answer, briefly explain how the technique works.
Preimplantation Genetic Diagnosis (PGD). April would be given hormones to make
her superovulate, and her eggs would be harvested from her ovaries. The eggs would
then be fertilized in a petri dish with Anthony's sperm and allowed to grow to the 8cell stage. At this point, a single cell would be removed from the embryo and would
be tested to see if it was TT (a normal, non-carrier for Tay Sachs), Tt (a carrier for
Tay Sachs), or tt (Tay Sachs). Only embryos that tested TT would be transferred
back to April's womb for gestation, ensuring that she would have a baby without Tay
Sachs who also did not carry the mutant allele.
*21.
(4 pts)
The concordance value for childhood chicken pox was 87% of MZ twins and 89% for
DZ twins in a large University of Minnesota study performed on twins reared
together. How would you interpret these results? Is childhood chicken pox primarily
influenced by genes or the environment? Explain.
When the concordance values for a trait are essentially the same in MZ and DZ twins,
it is almost certainly under environmental control, not genetic control. This is
especially true when the twin pairs used to establish the concordance values were
reared together. In this case, chicken pox is highly infectious, and if one twin gets the
virus the other twin is very likely to get it as well. According to the concordance
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BIO 2 Laboratory
Spring 2008
values, there is close to a 90% chance that if one twin gets the virus the other will
also catch it.
*22.
(6 points)
Fill in the blanks.
1.
The peroxidase activity in heme breaks down hydrogen peroxide into
water and molecular oxygen.
2.
In the presence of iodine, starch turns a blue-black color.
3.
During embryogenesis, the Hensel twins shared two membranes, the
chorion and the amnion.
*23.
(6 points)
Draw a diagram showing how cell fusion can be used to clone mice.
Female mouse mates
with a male mouse and
becomes pregnant
The embryo is removed
from the mouse and the
cells are separated into
several different petri
dishes
Eggs are removed from
a second female mouse
and the nuclei are
removed from the eggs
Depending on how many clones are desired, several cells from the
embryo are chosen, and the nucleus from each chosen cell is
added to an enucleated egg from the egg donor
The eggs (now each carrying a diploid
nucleus from the same embryo) are allowed
to divide into embryos and are then
transferred into a female mouse (the borth
mother) for gestation and birth
Lots of baby mice that
are genetically identical
to one another (but not
to their parents)
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BIO 2 Laboratory
*24.
Spring 2008
(5 points)
Circle all of the human traits below that are NOT inherited according to Mendel's
predictions.
Eye color - multifactorial
Presence of freckles
Detached ear lobes
Hair type (curly, wavy, or straight) incompletely dominant alleles
IQ (intelligence) - multifactorial
Smelly urine after eating asparagus
Ability to roll one's tongue
Ability to taste PTC paper
Red-green colorblindness - X-linked
Skin color - multifactorial
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