BIOL 41 Foothill College Wk 4 Micro Dna Fingerprinting Lab Report The file has been attached below. Please help me. Thank you so much. This is for my micro

BIOL 41 Foothill College Wk 4 Micro Dna Fingerprinting Lab Report The file has been attached below. Please help me. Thank you so much. This is for my microbiology class. Biol 41 Spring 2020
Name_______________________________________
DNA fingerprinting Lab Report (15 pts)
In today’s exercise, you are going to use DNA fingerprinting to diagnose a disease in a
simulated virus outbreak. So far, the epidemic has been confined to students at a small college
town, but you need to hurry to identify the virus before it spreads any further. Based on
symptoms and epidemiological evidence, the virus is suspected to be BCV (Brain Cramp Virus),
CPV (Chronic Procrastination Virus), or MIOV (Microbiology Information Overload Virus.)
Fortunately, there is an antiserum available for each of these, and it’s up to you to determine
which antiserum is appropriate. Viral DNA samples have been rushed to your lab and
technicians have already digested the samples with restriction enzymes. Your job is to run an
electrophoresis gel on your sample and compare its fragment pattern with the three known
viruses.
Materials
For each group of four students:
1. 20 mL of 0.8% melted agarose for pouring gel
2. Buffer for electrophoresis chamber
3. DNA samples
a. Standard DNA fragments
(For size comparisons this sample consists of nine fragments of the following sizes:
23,130 bp, 9416 bp, 6557 bp, 3000 bp, 2322 bp, 2027 bp, 725 bp and 570 bp where bp
refers to the number of base pairs in the DNA fragment)
b. CPV (Chronic Procrastination Virus)
c. BCV (Brain Cramp Virus)
d. MIOV (Microbiology Information Overload Virus)
e. Unknown outbreak virus sample
4. Automatic micropipette for measuring 20 uL
5. Pre-made gel for micropipette practice
6. Loading dye for micropipette practice
7. Gel bed (casting tray) with rubber end caps and gel comb
8. Electrophoresis chamber with lid, cables and power source
Methods
Preparing the Gel Bed
1. Close off the end of a clean, dry casting tray using the two rubber end caps.
2. Place the casting tray into the electrophoresis chamber.
3. Place a comb in the first set of notches near one end of the gel bed. Make sure the comb sits
firmly and evenly across the bed.
4. Obtain a flask of liquid agarose from the 55°C water bath. Be certain your agarose is
completely melted and clear. If it has begun to solidify, re-melt it with a microwave or hot
plate and cool it again to 55°C. (Lumps in the agarose will inhibit the migration of DNA
fragments and agarose that is too hot will melt the casting tray.)
5. When the agarose has cooled to 55°C, pour the agarose into the casting tray.
6. Let the agarose harden in the casting tray on a level surface until it’s completely solid (usually
about 20 minutes).
7. After the gel has completely solidified, gently pour the electrophoresis buffer into the
electrophoresis chamber. The gel should be oriented so that the sample wells are nearest the
negative (black) electrode terminal. (Why?)
8. Slowly and carefully, remove the comb. (Your instructor will demonstrate.) Once the comb
has been removed, you are ready to load your DNA samples.
Gel Loading Practice
1. Before pipetting, gently tap the microfuge tube on the counter until all the liquid is collected in
the bottom of the tube.
2. Carefully withdraw 20 l from the loading dye solution, and prepare to insert the sample into
the first well. It’s very important not to damage or even touch the sides of the wells with the
pipet, because if the edges of the well are not straight and intact, the quality of your DNA
bands will be diminished.
a. In order to stabilize your pipet as you load the gel, you should be seated with both your
elbows resting on the bench top. Hold the pipet in your dominant hand and use your
other hand to steady the pipet.
b. Carefully dip the pipet tip into the center of the well (without touching it) and gently
eject the sample into the well.
3. Continue practicing with the pipet until you feel comfortable with this technique. You can
rinse the dye from the wells and reload them as often as you like.
Loading DNA Samples and Running the Gel
1. Heat the DNA samples for two minutes at 65°C. After heating, place the samples immediately
on ice until you load them into the gel. (The DNA samples were previously digested with
restriction enzymes that leave DNA fragments with single stranded end pieces called, “sticky
ends.” These sticky ends might have joined back together with their complementary sticky
ends before the lab began, so heating causes the sticky ends to separate once again.)
2. Load each DNA sample into the wells in consecutive order. Each well should receive 20 l of
DNA sample. Make sure to use a new tip for each sample.
3. Make sure the power supply is unplugged and turned off. Snap the chamber lid in place with
the electrode terminals. The negative (black) wire must be connected to the negative terminal
and the positive (red) wire must be connected to the red terminal.
4. Plug in the power supply and turn it on. Set the voltage to the setting recommended by your
instructor.
5. If everything is working properly, tiny bubbles should rise up in the buffer solution at either
end of the electrophoresis chamber.
DNA Fingerprinting Protocol
6. After a few minutes, you should be able to see the blue dye migrating across the gel. DNA
fragments will be migrating behind the dye. In order to insure the DNA fragments have been
separated out into distinct bands, you should let the dye move 4-7 cm away from the wells.
Staining DNA with InstaStain™ Sheets
1. The best stain for DNA gels is ethidium bromide (EtBr.) EtBr bonds to DNA between the base
pairs and fluoresces when exposed to UV light. However, EtBr is a mutagen so it is essential
to wear gloves at all times when handling it.
2. After electrophoresis is completed, lift the casting tray out of the electrophoresis chamber.
Slide the gel onto a sheet of plastic wrap on a flat surface. Moisten the gel with a few drops of
electrophoresis buffer.
3. Wearing gloves, place the unprinted side of the DNA InstaStain™ sheet on the gel. This sheet
contains the EtBr stain.
4. Firmly run your gloved finger over the entire surface of the InstaStain™ sheet. Do this several
times.
5. Place the casting tray and a small beaker on top of the InstaStain™ sheet. This will ensure that
the stain in the sheet makes good contact with the gel surface.
6. After 2 minutes, remove the InstaStain™ sheet. Place the used sheet in a zip-lock bag and
dispose of it according to your teacher’s instructions.
7. Transfer your gel to a short wave (300 nm) UV transilluminator for viewing the DNA bands.
Be sure to wear UV protective goggles!
DNA Fingerprinting Protocol
Results
1. Draw your gel and DNA bands in the box below. Sample A is composed of seven standard
DNA fragments of known sizes in base pair (bp) lengths. These known bands can be used to
analyze the sizes of bands from our viral DNA. To the left of the box, label the standard bands
( 6751bp, 3652bp, 2827 bp, 1568 bp, 1118 bp, 825 bp, and 630 bp). Is the longest
standard DNA fragment closest or furthest from the wells?
DNA Ladder
CPV
BCV
MIOV
UNKNOWN
1
Based on the gel in the datafile and your drawing.
2. Can you tell which virus is responsible for the outbreak? If yes, which one is it? How did you
know?
3. How long is each virus in base pair units? Show your calculations. (By the way, real viruses
are typically much longer.)
a. CPV
b. BCV
c. MIOV
Discussion
1. When many restriction enzymes digest DNA they make a staggered cut that leaves
single-stranded ends called, “sticky ends.” Why are they called sticky?
2. Below is a diagram of pAMP, an ampicillin resistance plasmid from Escherichia coli. The
diagram shows the restriction sites for the endonucleases, EcoRI, BamHI and HindIII. If this
plasmid was exposed to all three enzymes and then run through an electrophoresis gel, how
many bands would you expect to see? Counting the bands and starting from the band closest to
the wells, which band would contain the ampicillin resistance gene?
Eco RI
Amp Gene
pAMP
Bam HI
Hind I
3. Restriction enzymes come from bacteria. Can you think of a function these enzymes might
have in bacteria?
4. What would be the effect on running the gel, if you mistakenly put water in the buffer
chamber instead of TAE?
5. What is the purpose of the buffer? and state what each component of the buffer do?
T = Tris HCl
A = Acetate
E = EDTA
6. What is meant by the phrase “DNA runs to red”

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