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DNA Isolation and Analysis Lab Report

Abstract:
Utilizing Drosophila as a model for human disease proved to be
an effective research tool. Seperating the plasmids from the cDNA
proved easy when using the QuickLyse purification system. By
isolating various genes in the Drosophila, researchers can pinpoint
specific genes which are commonly linked to diseases. This new
ability will allow for genetic mutation of disease carrying genes,
which will, hopefully, ultimately eliminate diseases such as cancer
and diabetes.
Introduction:
In the DNA isolation and analysis lab, the Drosophila was used as the
model for human disease. There are several reason behind using the
Drosophila as a model: 1.) they are small and easy to keep, 2.) they have a
short lifespan, 3.) they are a human homolog and 4.) they are easy to do
modifications to.
Drosophila homologs have been found for 70% of the known human
cancer genes and 60% of known genetic disorders involved in human
diseases. Drosophila models for human diseases address diseases such as
Type II diabetes, metabolic disorders, neurological diseases such as
Alzheimers, Parkinsons, Huntingtons, Fragile X, muscular dystrophy,
epilepsy and heart-related diseases such as cardiomyopathy. Drosophila
models for human cancers relate to human cancers such as glioblastoma,
salivary gland cancer, prostate cancer, ovarian cancer and leukemia (Rubin
2012).
In order to find a cDNA, a plasmid A and B cDNA was isolated. The
cDNA was then amplified and sequenced. A BLAST search was used to search
homologous human genes, protein domains and possible disease-related
function in humans with the Drosophila gene.
Using these homologous human genes it is possible to get a better
understanding of physiology and diseases in humans. It is much easier to
study (kill) fruit flies than humans just to understand their genes.

Materials and Methods:

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Experiment Part A:
1. Prepare an assortment of E. coli colonies containg
a plasmid with a gene for ampicillin resistance one
Drosophila cDNA sequence in an agar plate.
2. Pick two colonies and inoculate into Luria broth
and penicillin.
3. Grow overnight at 37C.
4. Store for six days at 4C.
Experiment Part B:
1. Using the two colonies chosen from part a, isolate
the plasmid DNA using the QuickLyse Miniprep
Plasmid DNA Purification System.
2. Mix cultures to resuspend the cells.
3. Pipet 1.5 mL of E. coli cell suspension into a
QuickLyse Lysis tube, make sure all tubes are
properly labeled.
4. Report with the second culture.
5. Centrifuge tubes for one minute at 13,000 rpm at
room temperature.
6. Pour off supernantant from tubes.
7. Add 400 ul of ice cold Complete Lysis Solution to
the pelleted bacterial cells.
8. Mix thoroughly by vortexing at the highest setting
for 45 seconds.
9. Incubate at room temperature for five minutes.
10. Decant the lysate to a labeled QuickLyse spin
column.
11. Centrifuge for one minute at 13,000 rpm.
12. Add 400 ul of Buffer QLW to the top of the
QuickLyse spin column to wash the column.
13. Centrifuge for one minute at 13,000 rpm.
14. Discard the flow-through.
15. Centrifuge for one minute at 13,000 rpm.
16. Transfer the spin column to a new labeled tube.
17. Discard the waste collection tube.
18. Add 50 ul of Buffer QLE directly onto the center
of the QuickLyse spin column and let sit for one to
two minutes.
19. Centrifuge for one minute at 13,000 rpm.
20. Discard the column and cap. The liquid
contains your plasmid DNA for PCR and DNA
sequencing.
Experiment Part C:
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1. Label two .2 mL thin walled PCR tubes with your

unique identifiers on the side.
2. Label one .2 mL thin walled PCR tube as negative.
3. Add 24 ul of PCR master mix to each of the three
tubes and gently cap them.
4. Add 1 ul of plasmid DNA to each of the two tubes
labeled with identifiers.
5. Add 1 ul of sterile water to the tube labeled
negative.
6. Load tubes into the PCR machine. Select
Programs then PlasmidPCR. The machine will
began to run.
7. After cycle is complete remove the tubes.
Experiment Part D:
1. Set-up the electrophoresis unit.
2. Weigh 250 mg of agarose and place in a 125 ml
Erlenmeyer flask.
3. Measure 25 mLs of 1XTAE buffer using a
graduated cyclinder and pour into flack.
4. Gently swirl to mix the agarose into the buffer.
5. Place the flask in the microwave for 40 seconds.
6. Remove flask and gently swirl.
7. Let the gel solution cool for about two minutes.
8. Add 1 ul of ethidium bromide dye to molten
agarose and gently swirl.
9. Cast gell into gel tray.
10. Allow to harden for ten minutes.
11. Remove dams.
12. Pour electrode buffer (1XTAE) into the unit.
13. Remove the comb.
14. Obtain plasmid DNA samples.
15. On a piece of parafilm, mix 2 ul of plasmid
DNA, 2 ul of 6X loading dye and 8 ul of water on
two different spots and mix gently (one for
Plasmid A and B).
16. Load 5 ul of the DNA ladder as a molecular size
standard into the first well.
17. Place 4 ul of 6X loading buffer into each of the
PCR tubes, gently pipet up and down to mix.
18. Load samples as follows: Well 1: PCR DNA
Ladder, Well 2: plasmid A DNA, Well 3: plasmid A
PCR product, Well 4: plasmid B DNA, Well 5:

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plasmid B PCR product and Well 6: PCR negative

control.
19. Insert electrodes into the power supply and
turn the power to 100 volts.
20. Once the bromophenol blue tracking dye has
migrated about of the length of the gel, turn
the power off and remove the gel.
21. Place the gel on UV box in the middle of the
blackened glass.
22. Set hood with camera on the box.
23. Turn on UV light, zoom with camera and take
the photo.
24. Save the image to the computer.
Experiment Part E:
1. Use the dideoxy method to sequence the DNA.
Results:

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Discussion:
For both cDNA sequences, BLAST was used to identify the protein. For
the cDNA sequence A the accession # is NM-165678 and the protein ID # is
NP-724798.3. For the cDNA sequence B the accession # is AM711972 and
the protein ID # is CAN84669.1. The human homologue ID number is
NA_005914. The observations for each of the six lanes in the gel are as
follows:
1.) There is an insert band in the lane.
2.) Insert band is needed larger than 206p.
3.) Insert band is 506b.
4.) Insert band A seems to fluoresce more than B.
5.) B has least secondary binding.
6.) DNA in plasmid DNA.
Literature Cited:
Bio 230W Lab Manual for DNA Isolation and Analysis.
Rubin, G.M., 2012. HHMI News. http://www.hhmi.org/news/rubin3.html
Pandey, U.B., & Nichols, C.D., 2011. Human disease models in Drosophila
melanogaster and the role of the fly in therapeutic drug discovery.
Pharmacological Reviews, 63, 411-436.