Mark Pipp, Matt Boyle, Ryan Lewis, Connor parrell

Dr. Knittle
AP Biology- 6th hour
May 27, 2015
Molecular Biology Lab Report
Introduction
DNA electrophoresis is a technique that is used to isolate sections of DNA that are cut by
restriction enzymes. To understand DNA electrophoresis it is necessary that we understand
restriction enzymes and where they came from. Restriction enzymes come from bacteria which
use these enzymes as a crude immune system to defend against viruses. This defense is
accomplished due to the ability of these enzymes to cut apart the sugar phosphate backbone of
DNA. This cut is done with two incisions that leave two strands of DNA. Two strands are created
from the one bacterial chromosome which is circular in structure. Each of the different restriction
enzymes cuts DNA in different locations as an evolutionary advantage, in order to combat the
rapidly changing viral DNA . Different bacteria can pick up different enzymes through bacterial
transformation and bacterial conjugation.
DNA electrophoresis uses the properties of restriction enzymes in the technique. Sections
of DNA are cut with different restriction enzymes to isolate genes. Genes can be detected by
their size. Approximate sizes of DNA strands are found by calculating the size of an individual
base pair multiplied by the amount of base pairs in the gene. One drawback of this procedure is
that you must know how long the gene is before the technique is carried out. However, once the
approximate length is determined the process is a breeze. If you accurately replicate these steps
the desired result will work as planned. Once you are finished these genes, once isolated, can be
genetically engineered into various organisms.

Procedures
Digest Procedure
1. Label four microtubes H, E, L, and P. They stand for Hind III, EcoR I, Lambda
DNA, and Pst I respectfully.
2. Setting the micropipette to 6 l, add 6 l of Lambda DNA to each tube.
3. Setting the micropipette to 9 l, add 9 l of the restriction buffer to the H, P, and
E tubes, then add 11l of the buffer to the L tube.
4. Add 2 l of Pst I, EcoR I, and Hind III to their respective tubes, replacing tips
between each tube.
5. With each tube secured shut, thoroughly mix the reagents in the tubes, then,
spaced evenly, put the tubes in a microcentrifuge and pulse spin them. Only place two in
at a time to make it evenly weighted.
6. Leave tubes in a tube holder and allow 48 hours at room temperature to incubate.
Gel Prep
1. Carefully open the gel box and tightly insert the wells in their slots, making sure
they are firmly in place.
2. Make sure that the casting tray is in place between the wells. Taking the 8-Bit
comb, place it on the channel, not in the slots, that will make your gel too thin, place the
comb on the other side, standing on the pegs. The comb has to be perpendicular and level.
3. Pour 60 ml of 1x TAE into a 250ml glass beaker and mix in 0.6 grams of agarose
in.
4. Heat up the solution until agarose is completely dissolved. Microwaving for 15
seconds at a time is suggested. (Be careful, as this will be very hot, use oven mitts)
5. With dams securely in place, carefully pour the solution in the gel box, between
the dams. Be careful not to disturb your comb
6. Make observations as the gel cools and hardens.

7. After about 25-30 minutes, carefully remove your comb, and the wells.
8. With a finger holding the gel in place, lift the whole casting tray out and very
carefully set it on the table.
9. Get a thick piece of plastic, and slide the gel on to it. Obtain a plastic bag, and
pour a bit of TAE into it
10. GENTLY slide the gel into the bag and close it. set this aside for when the DNA is
done incubating
Loading/Running The Gel
1. After the 48 hours, take your H, E, L, P microtubes and insert 2 l, using your
micropipette, of loading dye. Make sure to switch tips between every insertion to avoid
cross contamination.
2. Grab a fifth microtubule, label it M, and put 11 l of the Marker into it, this is for
a frame of reference for the bands of DNA. Insert 2 l of loading dye into this tube.
3. Mix and microcentrifuge all five tubes thoroughly.
4. Remove the dams from the gel box
5. Obtain your gel, and carefully set it in the casting tray.
6. Set this in the gel box, with the wells facing the negative cathode
7. Pour enough TAE into the box so that it just covers the gel.
8. Pipette 15 l from each tube into the wells, replacing the tip after every one. Keep
a small amount of pressure on the plunger of the pipette so you don't make any air
bubbles.
9. Keep track of which well is which. It may be helpful to put it in this order; H-E-LP-M
10. Put the cover on the box, and attach the anodes and cathodes. Plug it in and set it
to 120 volts.
11. You will see a blue and purple lines appear, stop the electrophorese when the
purple line is at the bottom of the gel. This will take roughly 30 minutes.
12. Remove the gel and casting tray and place into a gel staining tray with 120 ml of
Fast Blast DNA stain
13. Mark the tray with your hour and name. Leave overnight to stain.
Analyzing Results

1. After staining, take your gel and carefully transfer it to an overhead. Turning on
the overhead, you will see your bands of DNA.
2. To make things easier, place a clear overhead sheet over the gel and carefully
trace the DNA lines
3. Measure how far the DNA has moved from the wells with a ruler on the overhead
sheet.
Observations
Liquid gel was microwaved for extra 15 seconds
After initial pour no leaks or problems seen
Small hair like thing on gel
2:19 gel is becoming slightly cloudy
Shlarein lines visible
TAE moving around electrodes
More bubbles visible on the negative end
Dye in separate places and starting to spread and turn purple
Air fogging the gel
Some gel from previous class in bottom of the negative end
Purple dye is farther than the blue dye
No DNA in P column of the gel
Top of gel broke off where the wells were
Results
The data shows the first well having a very high amount of base pairs normally over 15,000 but
then dropping significantly to below 10,000. The base pair amounts then start to lower at a

slower rate. The line of best fit was also much closer to the bands that were farther away from
the wells. The r value for the line of best fit was .826.
Conclusion
Electrophoresis/DNA fingerprinting is used to determine the size of fragments cut by certain
restriction enzymes. These restriction enzymes cut only at their specific protein recognition sites.
This is useful due to the fact that no two restriction enzymes code for the same recognition site,
which allows for a fingerprint like uniqueness that is plausible only with ones DNA. Given
the collected data from the electrophoresis experiment, other sizes of parts can be hypothesized.
You can do this by following the size of the base pair to the line of best fit made on the log sheet.
This data tells you approximately how many millimeters the base pair would probably go if
permitted the same circumstances.

Questions
4. For which fragment sizes was your graph most accurate? For which fragment
sizes was it least accurate? What does this tell you about the resolving ability of agarose-gel
electrophoresis?
The graph was the most accurate for the 5th fragment and least accurate for the first fragment
size. This tells us that the resolving ability of agarose-gel electrophoresis becomes more accurate
as more bands are measured.

1. Discuss how each of the following factors would affect the results of
electrophoresis:
a. Voltage used- Higher voltage would lead to faster movement of
the bands of DNA, and lower voltage would lower the speed
b. Running time- Longer running time would lengthen the distance
between bands, and could cause some bands to fall off. Shorter run times would
lead to mixing bands and unreadable data
c. Amount of DNA used- If more DNA had been used, the bands would
have been darker because more of the fragments would have traveled the same distance
in the gel. The bands would only have been more distinct and distinguishable.

d. Reversal of polarity- Reversal of polarity would cause the bands

to run off the gel very early on.
2.

Two small restriction fragments of nearly the same base-pair size appear as a single

band even when the sample is run to the very end of the gel. What could be don't to resolve
the fragments? Why would it work?
You could fix this by increasing the length of time and decreasing the voltage of the electrophoresis. This
would give the DNA longer to run, but not let it run off the gel. Its akin to zooming in on a graph to see
the difference between two points.
1

What is a plasmid? How are plasmids used in genetic engineering?

Plasmids are small rings of DNA. They are used in genetic engineering because it is easier to manipulate
them into taking up genes than it is to change the whole DNA sequence of the cell.
2

What are restriction enzymes? How do they work? What are recognition sites?

The enzymes are endonucleases that cut the Phosphate backbone bonds of the DNA. They only cut at
specific proteins, the recognition site.
3

What is the source of restriction enzymes? What is their function in nature?

They occur naturally in prokaryotes and are used to cut up invading viral DNA that happens to get
through the cell wall and plasma membrane of the bacteria.

Describe the function of electricity and the agarose gel in electrophoresis.

The electricity is used to pull the DNA towards the positive electrode so that it will separate. The gel is
helpful because it is slightly porous, allowing the DNA to flow through it, but at varying speeds
depending on the size of the fragment.
5

If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4000, 2500,

2000, and 400 base pairs, sketch the resulting separation by electrophoresis. Show starting point,
positive and negative electrodes, and the resulting bonds.

Base

4000

2500

2000

400

What are the functions of the loading dye in electrophoresis? How can DNA be prepared for

visualization?
The dye allows the DNA to be more distinct so that accurate measurements can be made in determining
the distance traveled and the amount of bands.
7

Use the graph prepared from the lab data to predict how far (in mm) a fragment of 8000

base pairs would migrate.

A piece of DNA of that size would probably run about 17.5 millimeters
8

How can a mutation that alters a recognition site be detected by gel electrophoresis?

If you ran the normal and the mutant at the same time, you could see the change in the mutant band,
because the mutant bands would be longer. This would be due to the fact that, because of the mutation,
the recognition site wouldnt be cut.
Page 38

What evidence do you have that each enzyme cuts the DNA at different locations?

Because we started with four samples of the same DNA, the evidence is the difference in band
length. If it was cut at the same locations, the bands would all be the same.

When this activity has been completed, describe what you have done in no more

than two sentences.

We used three different enzymes to cut DNA and compared the base pair length of the fragments.
We also used totally rad micropipettes too, You da best DK

Compare the two methods--Direct gel examination and semi-log graph-- of

determining the fragment size (in base pair units). Which method seems to be more precise?
Explain your answer.
Direct gel examination because in the semi log graph method you are making estimates based on
the distanced traveled by a control group. However if an error occurs in the experiment and throws off
this group then all of the data is thrown off because the rest of the fragment sizes are estimated by this
group. In Direct gel examination if one group has incorrect data due to an error but the rest are still
correct you can still get correct data for these groups since you are measuring them directly.