A University of Texas Research Experience

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A University of Texas Research Experience

• A Journal of a Teachers Experiences
• By Joy Killough

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The Beginning
Near the beginning of the 2003-4 school year I
received an e-mail advertising a program at the
University of Texas at Austin. The program would
connect teachers with professors at the
University and give the teachers the opportunity
to be involved in real scientific research. This
is an account of my activities during this time
and is one of many possible realities in an
academic research setting. It was difficult,
demanding and frustrating and yet, a wonderful,
rewarding experience that brought about great
personal growth.
The turtles outside the Biological Laboratories
building. A great place to ponder problems.
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Applying to RET
Research Experience for Teachers is a national
program funded by the National Science
Foundation. Placement in the UT program was
competitive and required a lengthy application
with references and interview. The interview
marked the beginning of my association with the
University of Texas at Austin. One purpose of
this project is to make university resources more
accessible to teachers and realizing this goal
starts with something as seemingly simple as
being able to park and find your way around
campus. The parking issue was a big concern of
mine as Austin area lore has it that parking is
impossible at UT. This myth was quickly
debunked through familiarity and I eventually
found the closest campus garage.
Click here for a Map of the UT Campus
This flower is from the pond outside the lab.
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Selecting a Project
After my selection as an RET participant, Dr. Jay
Banner and Dr. Nelson Guda worked to match my
interests and abilities with the projects and
needs of the researchers willing to host an RET
teacher. I interviewed with two professors in
the Integrative Biology Section. Both of the
professors were willing to work with me, which
meant a tough decision because they were both
working on interesting topics. I chose to work
with Dr. C. Randal Linder because I had more
personal interest in the topics and techniques
involved in the cave DNA project.
A cave pool inside Inner Space Caverns in
Georgetown
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The Linder Lab Group
I began my work in the Linder lab by joining Dr.
Linder and his students during his weekly
seminars. A current paper pertaining to the
research interests of the lab would be suggested
by either Dr. Linder or one of the students
(graduate or undergraduate) and we would meet for
discussion.
The lab group was very easy to work with. I
probably drove them crazy with my questions about
everything and anything but they were unfailingly
cheerful and helpful. Lab meetings were informal
but informative.
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Safety Training

• Before beginning in the lab I was required to
  complete 4 safety trainings. The first of these
  was hands on Fire Extinguisher Training. I also
  completed two on-line classes dealing with
  laboratory safety and hazardous waste. These
  courses ensure everyone working in labs at the
  university has basic lab safety skills and
  understands hazards they are likely to encounter
  and well as understanding university procedures
  for environmental protection. One on one safety
  training was also required and provided for the
  Linder lab.

Waller Creek runs through the UT campus.
Following regulations helps to protect this
ecosystem.
From the Collections of the Texas Memorial
Museum, The University of Texas at Austin.
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The Research Problem
The research problem I worked on was a tiny part
of a very large puzzle. The big puzzle was
looking at the possibility of ancient DNA being
trapped in cave formations. This very exciting
prospect has the potential to add significantly
to knowledge in both geological and biological
arenas.
Before sampling any ancient cave formations the
modern system required analysis. I worked on the
characterization of microorganisms found in cave
drips and pools in three Texas caves, Inner
Space, Caverns of Sonora, and Natural Bridge
Caverns. I also helped set up a sampling system
for soil above the drip sites. The correlation
between the soil bacteria and the cave drip water
bacteria provided data for testing hypotheses
regarding flow types seen in these caves as well
as other information.
Collecting from a cave drip
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My Collaborator
Early in my work I met Katie Golder. Katie and I
would spend the summer together trying to solve
one puzzle after another. Katie had spent the
previous semester extracting DNA from water
samples collected from caves. These were the
samples we worked with through the summer.
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The Research Begins
Katie spend many hours in the spring training me
on the various techniques needed for our
investigation. Here she is working under the
laminar flow hood. Much of our work was done
here to prevent contamination of our bacterial
samples from the cave. A laminar flow hood has a
constant stream of filtered air flowing out,
providing an area free of airborne particles and
microorganisms.
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Preparing Extraction Equipment
Additional samples from the caves were collected
during the time I worked in the lab. Katie and I
prepared the equipment needed for the sterile
collection of water samples from cave drips and
pools within the three caves. Here I am cleaning
a disk, which holds a filter, in preparation for
autoclaving.
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Preparing DNA Extraction Syringes After cleaning,
the disks are reassembled under sterile
conditions, wrapped and autoclaved.
An autoclave in Biological Laboratories
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Data Collection in Inner Space Georgetown, Texas
In the caves, multiple sites are surveyed each
month. In the picture at the upper left, a
graduate student is recording the drip rate of
one of the DNA test sites. At bottom left a
glass plate that has been in place for a month is
retrieved. The plate will be analyzed in Dr.
Banners lab for calcite growth. Site
information collected includes temperature,
atmospheric CO2, and chemical tests on the cave
waters. Additional water samples are collected
and brought back to the lab for a series of tests.
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Collecting from a slow drip

• In the case of a slow drip site a sterile
  container is left in place until enough water is
  collected to run through the filter apparatus.
• After removing the container a new glass plate is
  placed.

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Attention to Detail is Critical

• The plate is rinsed, and packed. At each site
  careful attention is given to recording
  information.

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In the Cave
Some of the water samples for DNA extraction were
from standing pools as opposed to the drip
sites. The water is passed through a filter and
labeled carefully for later identification.
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Sample Collection

• Some of the drips are easy to collect from while
  others require quite a stretch.
• Amber steps on plastic to protect the delicate
  formations from body oils.

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Protecting the Samples
After the collecting, the samples are frozen in
liquid nitrogen for the ride back to UT. They
will be stored in a -80 degree freezer in the
lab. The field equipment carried down in the
cave must be repacked.
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Collecting the Soil Samples

• It was a scorching July day at about 100 when we
  set out to collect soil from above the drip
  sites. Katie and I had worked out a protocol
  which utilized concentric circles 1,3,and 5
  meters out from the sites. We collected two
  samples per circle at randomly located angles
  predetermined at the lab. Dr. Jay Banner,
  director of ESI, and Katie are marking the
  circles and preparing equipment.

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Sample Removal and Sterilization

• Dr. Jay Banner located the first site with
  GPS. It was on a very steep, overgrown incline
  of an overpass on I35. The thistles were
  especially difficult to work around. Here Jay
  and I are removing a soil sample from the soil
  borer. This was difficult due the crumbly nature
  of the soil and the need for sterility. Between
  each of the six samples per site the borer was
  sterilized. Since we all wanted to avoid being
  on the evening news for setting the hillside on
  fire this required a trip down the hillside to
  the culvert to flame the borer. Did I mention it
  was 100 degrees?

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Soil Site 2

• The first site took several hours. After a break
  to rehydrate we moved across the highway to the
  second site. I think we were all relieved to be
  out of the direct sun and off of the difficult
  slope. The second site had to be better. We
  didnt count on the cactus. At least there was a
  fragment of shade and we were more practiced at
  the collecting.

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Soil Site 3
It was with great relief that site three was
level, free of cacti and mercifully shaded. By
now we had been collecting for hours in the
extreme heat. In this picture you can see the
tube with an antimicrobial agent and one with
distilled water that were used to rinse to borers
before flaming.
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Water Sample Storage

• The extracted DNA samples are stored in this
  -80C freezer to prevent degradation.
• This freezer box contains almost 100 samples of
  DNA extracted from the three Central Texas caves
  in the past 2 years. It is ready to use in PCR,
  Polymerase Chain Reaction, a technique that will
  make a tremendous number of copies in a a couple
  of hours.

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PCR Preparation

• The lab freezer contains more storage boxes, but
  these are filled with reagents. Everyone in the
  lab has a box to keep their materials in. Katie
  is putting the items we need for a PCR on ice
  including master mix and primers. We will return
  for the Taq polymerase at the last minute.

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PCR
Under the laminar flow hood we combine the
extracted DNA, DNA nucleotides and primers, small
pieces of nucleic acids that base pair with the
region of DNA we are amplifying. One of the more
tedious parts of the lab work involves aliquoting
identical tiny amounts of reagents into as many
as 96 tiny tubes. The microtubes will be loaded
into a thermocycler, a machine that runs a
program of alternating heating and cooling.
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Electrophoresis and Staining

• After amplifying, the PCR product is separated by
  electrophoresis using the equipment in the top
  picture. The DNA bands are separated based on
  their size.
• The DNA bands are not visible without staining.
  The gels must be submerged in a tray of ethidium
  bromide on a shaker for ten minutes.

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Visualization of Gels

• In order to see the bands they are exposed to UV
  light. The image is focused using the computer
  and printed. The printed record is taped in the
  lab book for future reference.

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Preparing for TRFLP and DNA Sequencing
Anneke, a graduate student in the Linder lab
shows us how to prepare a polyacrylamide gel to
analyze terminal restriction fragment length
polymorphisms (t-RFLPs) in our DNA
amplifications. Cleaning the plate and lining up
the reservoir for the comb is first.
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Pouring the Gel

• At right we are inserting the comb and injecting
  the polyacrylamide.
• Setting up the gel took some time. Anneke
  emphasized to us the hazards of the unpolymerized
  polyacrylamide and the importance of very clean
  plates.

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Polymerization
Ultraviolet light causes the polymerization of
the polyacrylamide. After a few minutes under
the lights the gel is ready.
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The BaseStation
The gel is loaded in the BaseStation, a DNA
sequencing machine. The computer will direct the
loading of the samples into the wells formed in
the polyacrylamide 8 wells at a time.
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Adding Buffer and Loading the Samples
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Results Peaks of different lengths
Each peak is a separate terminal fragment of
DNA. They are marked with a fluorescent
primer. The diversity of bacteria in the caves is
evident. The identity of the bacteria is the
next step through DNA sequencing. Our results
were presented in a student forum.
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What I learned this summer

• I learned a tremendous amount this summer.
  Scientific research is a lot more difficult than
  I imagined. I tell my students now you need to
  be very persevering to conduct scientific
  research. Dr. Linder was great to work with and
  I appreciate his letting me work on this project
  in his lab. He gave us a lot of latitude and
  expected us to be successful through our own
  effort, and he was always available for
  conferencing while happily helping Katie and I
  through many obstacles. We spent several weeks
  troubleshooting our PCR but in the end found out
  we needed to change the time in the thermocycler
  program to adjust to the change we made in
  primer. It was frustrating to lose that amount
  of time as we both had a goal and we were working
  within a bigger team that really wanted our
  results.
• Although I did not get as much experimental data
  as I would have liked, I gained a wealth of
  knowledge about science, the University of Texas,
  graduate school, biotechnology research,
  designing sampling techniques, caves,
  bioinformatics, and so on. In fact I could go on
  and on.
• This was a challenging but exciting experience
  that has positively influenced my teaching. I
  appreciate the sponsorship of NSF and The
  University of Texas at Austin, and in particular
  the help of Dr. C. Randal Linder and Dr. Jay
  Banner.