Plankton of Bamfield Inlet
A Molecular Ecology Survey
Celeste Leander, Science One
Fall, 2007
Introduction
The vast majority of biological diversity on the planet exists as microscopic organisms.
Most of these species are rare. In addition, biologists estimate that we probably cannot
culture 90% of microbes. That leaves us with a startling amount of biological diversity
that remains largely undocumented. So how do we know what’s out there if we can’t see
it, and we can’t isolate it?
Aplanochytrium sp. Members of this
genus are obligately marine. They are
ubiquitous decomposers, but are rarely
documented.
As our ability to access genomic information increases, we can use molecular technology
to survey unseen organisms. To do this, we collect a sample of living microbes from the
environment, extract DNA, and look at the sequence of a specific region of the genome to
identify the organism(s) in the sample.
We will spend several weeks evaluating the diversity of microscopic life collected from
the Bamfield Inlet. The first two steps (plankton tow and DNA isolation) will be done at
the marine station. The remainder of our survey will be done back at UBC.
Goals
One of the most important skills that you will begin to hone here, and in your
laboratories, is your ability to keep a lab notebook. Although bound notebooks are
typically desired, you can use a loose-leaf binder for this project. I will be giving you
handouts, so a binder will help you assimilate your information.
At the end of this unit, you will have learned how to isolate DNA, PCR a gene, run, stain,
and evaluate an electrophoresis gel, and perform a BLAST search. You will also learn
the theory behind fluorescent sequencing and molecular cloning. (These skills can be
added to your resume and will help distinguish you from other first-year students.)
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Contents:
Plankton tow and DNA isolation (at BMS)
Polymerase Chain Reaction
Gel Electrophoresis
Cleaning a PCR product
Molecular Cloning
Sequencing
Performing a BLAST search
Question:
What do you think is the most important skill to keeping a lab notebook?
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Handout 2:
A. Plankton Tow and morphological diversity
A plankton tow (or net tow) concentrates a sample of stuff that is floating or swimming in
the water. To do a plankton tow, we utilize a plankton net. Plankton nets come in a wide
variety of sizes. A plankton net consists of a long, fine mesh net, with a removable
container with a sieve at the cod end. As the net passes through the water (typically for a
few minutes), water passes through the net, and then the sieve, leaving the cells trapped at
the cod end. We will be using a net with a mesh size that will trap a variety of eukaryotic
cells. When the tow is finished, the sieve end of the net is quickly unscrewed and the
remaining water with concentrated cells is dumped into a collecting jar.
B. Isolation of DNA
DNA isolation is a series of steps that will crack open a cell wall (if necessary), and then
selectively get rid of everything that isn’t DNA (or RNA). There are many cheap ways to
isolate DNA, including using meat tenderizer, dish detergent, and ethanol (see
http://learn.genetics.utah.edu/units/activities/extraction/). These techniques work fine if
you have lots of relatively pure starting material. There are also standard, inexpensive
laboratory methods that researchers typically employ to isolate DNA. These often
require chemicals such as chloroform that should be used in a fume hood. Since we want
to extract DNA from a few cells with high efficiency in an open lab, these are not our
best options. We are lucky enough to have access to a streamlined kit that will greatly
enhance the efficiency of your efforts. The kit we are using is called Masterpure
Complete DNA and RNA Purification Kit by Epicentre. The directions below are
slightly modified from the directions that come with the kit.
1. Your first task is to concentrate the cells from your plankton tow even further. Using
a plastic pipette, fill one 1.5 ml epi-tube with water from the collection jar. Spin at high
speed in the microcentrifuge for 2 minutes. (WARNING: You must always balance a
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centrifuge. Use someone else’s sample, or a tube with water, if necessary. Failure to
do so will damage the centrifuge. Ask us if you don’t understand this!). Quickly
suck off all but about 25 µl of the water. (The swimmers will immediately start to
migrate up the water column, so work fast).
2. Vortex 10 seconds to resuspend the cells. Then add 300 µl of “Cell Lysis Solution
with Proteinase K” (Tube ‘A’).
3. Incubate at 65C for 15 minutes, vortex every 5 minutes.
4. Place samples on ice for 5 minutes.
5. Add 150 µl of “Protein Precipitate Reagent” (‘B’) to your tube. Vortex for 10
seconds.
6. Centrifuge at maximum speed for 10 minutes.
7. Transfer the supernatant (the liquid part) to a new tube. Discard the old tube with the
pellet at the bottom.
8. Add 500 µl isopropanol (‘C’) to the supernatant and carefully invert 30-40 times.
9. Centrifuge at maximum speed for 10 minutes.
10. Carefully pour off the isopropanol without disrupting the pellet.
11. Rinse twice with ethanol (‘D’), carefully pouring off each time without disrupting the
pellet.
12. Leave the cap open at room temperature overnight to evaporate any ethanol that is
left.
Questions:
From your observations, what do you think “Cell Lysis Solution” is made of?
What is it doing?
What is happening in step 3? Why does this require incubation at an elevated
temperature?
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The Polymerase Chain Reaction (PCR)
In the late 1980s, Kerry Mullis changed the face of biology with the invention of PCR
(polymerase chain reaction). Although Kerry Mullis’ paper was rejected from several top
journals, he went on to win the Nobel Prize in chemisty in 1993.
(http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html)
A typical eukaryotic genome contains thousands of genes and billions of nucleotides.
Amongst all that genetic soup, researchers are often interested in analyzing the sequence
of one tiny bit, typically a few thousand nucleotides. Because of this, almost any type of
genetic analysis requires lots of copies of a portion of DNA that the researcher is
interested in. PCR mimics DNA replication in a test-tube, and it specifically makes
copies of one selected region. This amplification of a piece of the genome, often copied
millions of times, results in the remainder of the genome becoming background noise to
an almost pure sample of copies of the amplified piece.
You will set up a reaction to amplify a region of DNA known as “small subunit
ribosomal DNA” or ssurDNA. As the name implies, this region of DNA codes for a
subunit of the ribosome. To perform a PCR reaction, a simple (expensive) hot block
called a thermocycler is utilized to rapidly change the temperature during the course of
the reactions. During tutorial you will figure out how this works.
The PCR reaction:
1. To a PCR bead (containing Taq polymerase, nucleotides, and buffer) add 10 µl of
water, 1 µl of primer mix (specific for ssurDNA), and 1 µl of your genomic DNA. Mix
by pipetting up and down a few times.
2. Place your tube in the thermocycler. Make note of the exact location (row and
column) of your tube.
Thermocycler schedule we are using:
Step 1: 95C 2 min
Step 2: 92C 45 seconds
Step 3: 48C 45 seconds
Step 4: 72C 1:30 min
Step 5: Go to 2, 37 times
Step 6: 72C 5 min
Step 7: 6C
forever
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Questions:
Do you expect to see triplet nucleotides coding for amino acids in your sequence?
Why or why not?
Why are we using ssurDNA as opposed to another gene?
How does PCR work? (Take notes from tutorial).
What is the significance of each temperature in the schedule in terms of what is going
on in the reaction tube?
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Electrophoresis
Hopefully, your PCR product consists of many copies of ssurDNA, and a small amount
of background DNA and RNA. Each piece of DNA is a long, linear, negatively charged
molecule. To isolate the ssurDNA from the other DNA, you will be loading and running
your PCR product in an agarose gel. (In the unlikely event that your PCR reaction failed,
this will become apparent as well.)
Agarose is a purified monomer of agar, which is an unbranched polysaccharide. Agar is
made by red algae where it is the primary structural component of the cell wall. It is
commonly used in cooking as a thickener of shakes, jellies, etc. We are using a 1%
agarose gel (by weight).
You will load your PCR products into a well at one end of the horizontal gel that is
submerged in buffer. To help see the DNA as you are loading it into the well, a dark blue
“loading dye” will be added. Once all samples are in the gel, a continuous electric
current will be supplied to the gel. Because DNA is negatively charged, it will run
through the gel towards the positive electrode.
Your ssurDNA is a predictable size of just over 1800 base pairs. To determine where this
DNA ends up on the gel, we will use a ladder. A ladder is a series of several dozen
pieces of DNA of known size that you can compare against your migrating DNA.
As your DNA migrates through the gel, the loading dye becomes diluted and will no
longer be visible. The finished gel must be stained before we can see your DNA. To
stain DNA, your gel will be soaked for 10 minutes in 5% ethidium bromide (EtBr). EtBr
intercalates between nucleic acids. After it has bound to DNA, EtBr fluoresces strongly
when exposed to UV light. (The reason for this is that the interior of the DNA strand is
hydrophobic. Water is a strong quencher of fluorescence, so EtBr does not fluoresce
strongly until bound.) Because EtBr binds strongly to DNA, it is considered a serious
mutagen, carcinogen, and teratogen. You will not handle EtBr in the Science One rooms.
However, your gel will be carefully transported to a lab after class where it will be
stained, viewed, and photographed under a UV hood. Successful PCR products will be
carefully cut out of the gel and placed in the freezer for next time.
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Question:
Lane 1 is a ladder. Lanes 2-5 are genomic DNA samples that were amplified with
one set of primers.
1. Which lane(s) (2-4) suggest that PCR worked for amplification of ssurDNA?
2. Lane 2 shows an intense narrow band at the top of the gel. What could this be?
3. Lane 3 shows a smear, but no distinct band. What could this be?
4. Why does lane 2 have several distinct bands?
5. Why do lanes 2, 4, and 5 have bands of different sizes?
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PCR clean-up
In order to proceed to the cloning step of our study, we must retrieve your ssurDNA out
of the agarose in which it is currently embedded. We will do this using a kit called
GeneClean. The kit comes with small glass beads and a salt solution. The idea here is
simple. You will melt your agarose band. Put in some glass beads and salt solution.
Negatively charged DNA will be temporarily bound to negatively charged silca with a
cation bridge. While the DNA is bound to the silica, the agarose will be washed away.
Then the salt will be removed to elute your DNA.
Modified GeneClean Protocol:
1. Add 150 µl of NaI solution to your band of agarose gel. Incubate at 55C to melt the
gel. Mix periodically until the gel is dissolved.
2. Add 20 µl if “Glassmilk” solution. Incubate at room temperature for 5 minutes,
mixing periodically.
3. Centrifuge for 30 seconds at high speed (14,000 rpm). Discard supernatant (the stuff
at the top).
4. Add 500 µl of “New Wash” (which is largely ethanol) to the beads. Swoosh the beads
up and down in the wash solution.
5. Centrifuge for 30 seconds at high speed (14,000 rpm). Discard supernatant.
6. Repeat steps 4 and 5.
7. Centrifuge beads for 1 minute (14,000 rpm) to dry pellet of ethanol.
8. Add 25 µl sterile H2O. Mix well to elute the DNA off of the glass beads.
9. Centrifuge for 30 seconds (14,000 rpm). Remove supernatant with DNA to a clean,
labeled test-tube.
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Molecular Cloning
At this point, you have isolated one gene (ssurDNA) from possibly thousands of
individual organisms that were in your initial sample. How many species would you
guess are represented in your tube? In order to sequence the gene you have isolated, the
genes from different organisms must be separated from one another. This is
accomplished by molecular cloning.
We will use special E. coli cells to do the job of molecular cloning for us. These cells
will take up one, and only one, strand of external DNA when exposed to an
environmental shock.
You will first incorporate your amplified strands of DNA into specially engineered
vectors. (What is a vector?) Once your PCR product is in the vector, the vector will be
mixed with competent E. coli cells, which will take up the entire circular unit of DNA.
The cloning vector is shown below:
The TOPO cloning vector has several genes, each providing an important protein
product.
Cloning protocol:
(Because of the sensitive nature of the living E. coli cells, cloning will be done for you in
the research lab.)
Prep: One vial of competent E. coli cells will be removed from -80C storage to a bucket
of ice. While the cells are thawing, the following three steps will be done.
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1. The TOPO® Cloning reaction is set up using the reagents in the
following order: 1-4 µl PCR product, 1 µl dilute salt solution, sterile water to a total
volume of 5 µl, 1 µl TOPO vector.
2. Mix gently and incubate for 5 minutes at room temperature.
3. Place on ice.
4. Add 2 µl of the TOPO cloning reaction to a vial of thawed E. coli cells and mix
gently.
5. Incubate on ice for 15-30 minutes.
6. Heat-shock the cells for 30 seconds at 42C without shaking. Immediately put tube on
ice.
7. Add 250 µl of room temperature SOC medium.
8. Incubate at 37C for one hour with shaking.
9. Spread 10- 50 µl (typically one at 10 µl, and one at 50 µl) of bacterial culture on a prewarmed agar plate containing X-Gal and 100 µg/ml of streptomycin. Incubate overnight
at 37C.
Next time, each group will choose one E. coli colony. Your lucky colony will be subcultured into broth media with antibiotics where it will grow over night at 42C. These
cultures will then be stored in the refrigerator for clean-up.
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Questions:
1. What benefit is there for a bacterial cell to take in external DNA when exposed to
environmental shock?
2. What is the function of the antibiotic resistance genes, and the lacZ gene?
3. Why is your PCR product inserted in the lacZ gene?
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Plasmid Miniprep
You now have loads of E. coli cells, each containing a copy of your chosen sequence.
The kit we will use today (Fermentes Gene Jet Plasmid Miniprep or other similar plasmid
miniprep kit) will isolate DNA from the E. coli cells, including the replicated vectors
with your PCR inserts.
Protocol:
1. Pellet 1.5-2 ml of cells. Remove liquid.
2. Add 250 µl Resuspension Solution (or equivalent) and vortex.
3. Add 250 µl Lysis Solution (or equiv.) and invert 4-6 times.
4. Add 350 µl Lysis Solution (or equiv.) and invert 4-6 times.
5. Centrifuge (max speed) 5 minutes.
6. Load the supernatant into the GeneJet spin column (or equiv.). Centrifuge 1 minute.
7. Add 500 µl Wash Solution (or equiv.), centrifuge 30-60 seconds. Discard flowthrough.
8. Repeat step 7.
9. Centrifuge empty column for 1 minute.
10. Transfer the column to a new epi-tube. Add 50 µl of Elution Buffer (or equiv.) to the
column. Let sit 2 minutes.
11. Centrifuge for 2 minutes.
12. Discard column.
You now have isolated DNA (including E. coli genomic DNA, plasmid DNA, and your
insert.) This DNA will be sent for sequencing at the campus central sequencing facility.
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Sequencing
The dideoxynucleotide sequencing reaction is very similar to a regular PCR reaction such
as the one you have already performed. The difference is that a small percentage of the
nucleotides are dideoxy- and fluorescently labeled according to the nucleotide base.
The result is an occasional incorporation of a dideoxy nucleotide, which terminates the
chain. As this process continues in the thermocycler, chains are made of varying lengths.
In fact, because so many chains are made during the course of the reaction, there are
likely to be many chains made that are terminated at each base.
When the reaction is finished, the product is run through a gel similar to the agarose gel
that you ran. However, the type of matrix used for sequencing is typically a
polyacrylamide gel.
As the product runs through the gel matrix, the shortest pieces will run off first. The
chains come off the end of the gel according to size, and the fluorescence is detected.
Each chain will fluoresce with the specific wavelength attached to the terminal
nucleotide. When pieced together, this gives the sequence of DNA nucleotides.
Question:
1. What is different about this reaction (versus a regular PCR reaction) that requires
a different gel matrix?
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BLAST
Since it takes special software to view and analyze the chromatograph that is generated
from the automated sequencer, I’ve asked the sequencing facility to provide us with just
the string of nucleotides inferred from your reaction. Be aware that it typically takes
many hours of critical evaluation of the chromatograph to come up with a sequence that
is strongly supported.
You will perform a BLAST (Basic Local Alignment Search Tool) search with your
sequence. The NCBI database (National Center for Biotechnology Information) is the
largest repository of genomic information in the world. You will submit your sequence,
and the BLAST program will search through the database and find the closest match(es).
Protocol:
1. Go to the NCBI homepage at http://www.ncbi.nlm.nih.gov/
2. Across the top bar, click on “BLAST”
3. On the left, click “nucleotide blast”
4. Cut and paste your sequence into the box at the top of the page
5. Click the “BLAST” button at the bottom of the page
6. Wait. During peak times, it may take several minutes to perform the search. (Lowest
usage times are typically in the evenings after the East Coast has gone home…)
When your results appear, scroll down until you see a table with accessions, description,
and various score columns. Look for the one with the highest “max ident” percentage.
(This should be near the top.) Some results will be of unknown sequences; you want to
find the highest match that actually has a name under the “description” column. When
you find it, click on the accession. Near the top of the resulting entry, there should be a
brief description of the organism (i.e. phylogenetic affinity).
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Questions:
1. What is your organism? (Taxonomic name)
2. What does it look like?
3. To which major group of eukaryotes does it belong?
4. What is its role in the ecosystem? (i.e. is it photosynthetic?)
5. What is its distribution?
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