Urban Microbial Community Dynamics and Metagenomics
Introduction:
Microorganisms are present in almost every natural environment and, as a group, they are often
the most numerous type of organism in an environment. Until recently, studies of microbial
diversity were limited to methods where only microorganisms whose growth could be detected
in the lab in nutrient media could be identified, quantified and studied. It is estimated that only
5% to 10% of the microbial diversity in any given sample can be cultivated in the lab and
detected in this way, and because of this much of the microbial diversity has gone undetected
and under appreciated. Recently metagenomic approaches have been introduced that detect a
much greater portion of the actual diversity in a sample. Metagenomic approaches are cultureindependent, which means that it is not necessary to be able to cultivate the microorganisms in
order to detect their presence. Instead, metagenomics requires isolating the genomic DNA (or
RNA in the case of some viruses) from the environmental sample being studied and using
“signature” DNA sequences as a proxy to quantify and characterize the microorganisms present
in the sample. Because these approaches are not selective for specific microbial genomes in a
sample the isolated DNA contains genomes representing, in principle, all of the microorganisms
that were present in the sample (hence the prefix “meta”, in metagenomics). When a sample is
being studied to determine the microbial population diversity and relative abundance in an
environmental sample, usually a specific
region of each microbial genome is
examined – in the case of bacteria this is
most often the gene coding for the 16S
rRNA.
The 16S rRNA gene is used
because it is present in all bacteria and it
has regions of sequence that are shared
among all bacteria as well as variable
regions in the gene that differ from species
to species.
Metagenomic approaches have now been
used to analyze numerous natural
environments across the globe as well as
the microbiomes of humans and a variety
of other organisms. However, urban
environments
have
largely
been
overlooked. Currently more is known about
microbial communities in the arctic and
distant regions of the oceans than is known
about the microbial diversity in any city –
despite the fact that a majority of the
world’s population now resides in cities
(Yergeau et al., 2010; Tringe et al., 2005). In order to address this lack of knowledge, in this lab
we will employ a metagenomic strategy, coupled with next generation sequencing, to analyze
the microbial communities present on streets, sidewalks and subway platforms in Brooklyn (in
and around the BC campus area). Depending on how the experiments are set-up the
metagenomic approach can be used to analyze any microorganisms from the environment,
however, for the work in this lab we will begin by focusing our investigation on the bacterial
members of the urban microbial community and leave viruses, fungi and other groups
microorganisms to future studies. The primary objective is to determine the bacterial
diversity and relative species abundance found in common urban environments. A
second important objective is to examine how these urban bacterial communities change
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Urban Microbial Community Dynamics and Metagenomics
over time. This is important work as urban microbial communities are likely to serve as a
reservoir for a number of human pathogens.
Outline of experimental work:
In order to investigate the urban bacterial
communities of Brooklyn you will collect
samples from the environment and
prepare and analyze these samples in the
microbiology lab. Initially you will work
with your instructor and the other
members of your group to identify the
site(s) to be sampled. The samples can
be collected using a simple swabbing
technique.
Each of your samples
contains the genomic DNA from all of the
bacteria that were isolated during the
swabbing process. The genome of each
bacterial
species
is
represented
proportionally to the number of bacteria of
that species that were collected on the
swab. For example, a species that makes
up 20% of the total bacteria isolated from
the environment on the swab will have its
genome represent ~20% of the total
number of bacterial genomes isolated.
After collecting the environmental sample
the bacteria need to be released from the
swab and the DNA from the bacteria
needs to be purified away from the
bacterial cells and all the other unwanted
dirt and debris that is picked up during the
swabbing step. Once the metagenomic
DNA sample has been purified it must be
quantified. We will use universal PCR primers to amplify a variable region within the bacterial
16S rRNA gene. The universal primers are designed to be variable in several positions (that is,
a given position in the sequence of the primer could be A, C, G or T for example, and the
resulting primers will not all be identical in sequence). The degenerate primers permit the
amplification of 16S rRNA gene from as broad a range as possible of the bacterial species
represented in the sample. Following PCR amplification the product can be cleaned up using
the QIAquick PCR Purification Kit (Qiagen). Each PCR sample will be visually examined by gel
electrophoresis to confirm the correct band size and each sample will be quantified using a
NanoDrop 2000 spectrophotometer. This amplified 16S rRNA gene region (referred to as the
amplicon) can then be cleaned-up, quantified and sent out for next generation sequencing. The
company that sequences our DNA samples uses the Roche/454 DNA pyrosequencing
technology (see animation here: http://454.com/products/technology.asp). The sequence data
from the 16S rRNA genes can them be compared to a database to determine the bacterial
diversity of the sample. These steps are listed here and detailed protocols for each procedure
are described below.
1. Identify sites to test and collect environmental samples
2. Isolate and purify metagenomic DNA samples
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Urban Microbial Community Dynamics and Metagenomics
3. Quantify metagenomic DNA
4. Amplify a variable region from the 16S rRNA gene from metagenomic sample using
universal primers
5. Analyze and quantify 16S rRNA gene amplicon
6. Clean-up 16S rRNA amplicon and send out for Roche/454 pyrosequencing.
Required background reading:
N. Pace review article: Mapping the Tree of Life: Progress and Prospects; MICROBIOLOGY
AND MOLECULAR BIOLOGY REVIEWS, Dec. 2009, p. 565–576
J. Handelsman review article: Metagenomics: Application of Genomics to
Uncultured Microorganisms; MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec.
2004, p. 669–685
Microbiology; A Human Perspective, 6th edition, Nester et al., chapter 1 and chapter 11 (or
equivalent chapters from other introductory microbiology texts).
Recommended* background reading:
Logares, R., Haverkamp, T. H. A., Kumar, S., Lanzén, A., Nederbragt, A. J., Quince, C., &
Kauserud, H. (2012). Environmental microbiology through the lens of high-throughput DNA
sequencing: Synopsis of current platforms and bioinformatics approaches. Journal of
Microbiological Methods, 91(1), 106-113.
Metzker, M. L. (2010). Sequencing technologies the next generation. Nature Reviews Genetics,
11(1), 31-46.
Scholz, M. B., Lo, C. -., & Chain, P. S. G. (2012). Next generation sequencing and bioinformatic
bottlenecks: The current state of metagenomic data analysis. Current Opinion in Biotechnology,
23(1), 9-15.
Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next-generation sequencing
technologies for environmental DNA research. Molecular Ecology, 21(8), 1794-1805.
* These reading cover next generation sequencing technologies and metagenomics data
analysis. They can be technical, but reading them to get the general sense of things can be
very useful.
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Urban Microbial Community Dynamics and Metagenomics
Protocol:
Step 1 and Step 2
1. Sample collection
a. Select sites for sampling
b. Swab from 4 spots within a square meter at the chosen sites
i. Wear gloves and take precautions to avoid contaminating the site or the
swabs from your skin or hair, etc.
ii. Dip a sterile swab into sterile swab solution (0.15M NaCl, 0.1% tween)
and then swab an area the size of your palm, or a little larger (~8x8 cm)
iii. Place the swab in a sterile 15 ml Falcon tube. You may need to break off
the end of the swab to fit it in the tube.
iv. Repeat this with three more swabs in three nearby spots next to where
you collected your first sample. Note – collect ALL of your samples from
within the 1 m2 space but do not re-swab any area. After swabbing place
each swab in the same 15 ml Falcon tube.
c. Back in the lab - release sampled material from swabs by adding 2 ml of swab
solution to the Falcon tube containing the 4 swabs and vortexing for 1 minute.
d. Remove each swab from the tube, but squeeze out all excess liquid on the side
of the tube.
e. Check the volume remaining in the 15 ml Falcon tube, it will probably be between
0.5 and 1.5 mls remaining in the tube and it should look like dirty water with
particulate debris that came along on the swab. Split the volume in the 15 ml
Falcon tube equally between two sterile microcentrifuge tubes. Take note of
approximately how much volume is in the microcentrifuge tube. Spin for 3
minutes at 14,000 x g (gravity is the same as relative centrifugal force; RCF).
This step should pellet all the debris and bacteria (and bacteria bound to debris)
into the bottom of your microcentrifuge tube. Make sure to face the hinge of the
tube to the outside (away from the center of the rotor – this is so any pellet will
collect at the bottom or side of the tube underneath the hinge). While your tubes
are spinning, calculate how much supernatant should be removed from your
tubes to leave ~100 ul remaining in each microcentrifuge tube.
i. Remove the calculated amount of supernatant using a pipette and being
very careful not to dislodge the pellet. When you are done you should
have ~100 ul of liquid (about a tear drop) left in the tube with the pellet of
debris and bacteria.
ii. Resuspend the pellet in the remaining ~100 ul of supernatant by briefly
vortexing or flicking each of the two tubes.
f. Follow the Mo Bio Power Soil kit (#12888) protocol for isolation of genomic DNA
form your sample. The steps will be listed here, but are essentially identical to
those written in the Mo Bio Power Soil protocol.
1. Transfer the entire resuspended ~100 ul from each of the two
microcentrifuge tubes to a single Power Soil PowerBead tube.
You are using the combined ~200 ul you collected from the swabs
instead of a soil sample. What’s happening: After your sample
has been loaded into the PowerBead tube the next step is
homogenization and lysis. The PowerBead tube contains a buffer
that will (a) help disperse the debris particles, (b) begin to dissolve
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Urban Microbial Community Dynamics and Metagenomics
2.
3.
4.
5.
6.
7.
8.
any humic acids that may be present and (c) protect the nucleic
acids (genomic DNA) from degradation.
Gently vortex or invert PowerBead tubes to mix. What’s
happening: gentle vortexing mixes the components in the
PowerBead tube and begins to disperse the sample in the
PowerBead solution.
Obtain solution C1 from your instructor. If solution C1 has a
precipitate in it then notify your instructor – it may be necessary to
heat the C1 solution to 60 C. What’s happening: Solution C1
contains SDS and other disruption agents required to complete
cell lysis of the bacteria in your sample. In addition, SDS is an
anionic detergent that breaks down fatty acids and lipids
associated with the cell membrane of many bacteria. If it gets
cold it will form a white precipitte in the bottom of the tube. If this
is the case then heating to 60 C will dissolve the precipitate of
SDS and the C1 solution can then be used.
Add 60 ul of solution C1 to your PowerBead tube and invert
several times to mix or vortex briefly.
Secure PowerBead tube horizontally using the vortex adapter or,
alternatively, use the TissueLyzer. Vortex for 10 minutes at
maximum speed (or for Tissuelyzer, shake for 2x 5 minutes at 25
Hz, rotating tube adapter between the two 5 minute shakings).
What’s happening: the vortexing step is critical for complete
homogenization and cell lysis. Cells are lysed by a combination of
chemical agents from steps 1-4 and mechanical shaking. By
randomly shaking the beads in the presence of disruption agents,
collisions of the beads with the microbial cells will cause the cells
to break open.
Place your PowerBead tube in the centrifuge as shown by your
instructor. Make sure your tube is balanced against another
PowerBead tube (from another group) containing approximately
the same volume (if there is not another group’s tube to balance
with, then use a microcentrifuge tube containing water that weighs
the same as your PowerBead tube). Centrifuge your tube at
10,000 x g for 30 seconds at room temperature. CAUTION: be
sure not to exceed 10,000 x g or the tubes may crack.
Transfer the supernatant to a clean, labeled, 2 ml collection tube.
Note: expect between 400-500 ul of supernatant at this step. The
exact recovered volume depends on the absorbency of your
starting material and is not critical for the procedure to be
effective. The supernatant may be dark in appearance and still
contain some particulate debris. Subsequent steps in the protocol
will remove the particulate matter.
Obtain solution C2 from your instructor. Add 250 ul of solution
C2 and vortex for 5 seconds. Incubate at 4 C (in the refrigerator)
for 5 minutes. What’s happening: solution C2 is a patented
inhibitor Removal Technology (IRT). It contains a reagent to
precipitate non-DNA organic and inorganic material, cell debris
and proteins. It is important to remove contaminating organic and
inorganic matter that may reduce the DNA purity and inhibit
downstream DNA applications, such as PCR.
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Urban Microbial Community Dynamics and Metagenomics
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x
g. Make sure to face the hinge of the tube to the outside (away
from the center of the rotor – this is so any pellet will collect at the
bottom or side of the tube underneath the hinge).
10. Avoiding the pellet (there may not be a visible pellet), transfer up
to 600 ul of the supernatant (liquid) to a clean 2 ml collection tube.
What’s happening: the pellet at this point contains non-DNA
organic and inorganic material including cell debris and protein.
For the best DNA yields and quality, avoid transferring any of the
pellet to the collection tube.
11. Obtain solution C3 from your instructor. Add 200 ul of solution
C3 and vortex briefly. Incubate at 4 C (in the refrigerator) for 5
minutes. What’s happening: solution C3 is patented inhibitor
removal technology (IRT) and is a second reagent to precipitate
additional non-DNA organis and inorganic material, including cell
debris and protein. It is important to remove contaminating organic
and inorganic matter that may reduce the DNA purity and inhibit
downstream DNA applications, such as PCR.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x
g.
13. Transfer up to 750 ul of supernatant to a clean 2 ml collection
tube. What’s happening: the pellet at this point contains non-DNA
organic and inorganic material including cell debris and protein.
For the best DNA yields and quality, avoid transferring any of the
pellet to the collection tube.
14. Obtain solution C4 from your instructor. Shake to mix solution C4
before use. Add 1.2 ml (2x 600 ul)of solution C4 to the
supernatant, being careful not to overflow the tube). Close tube
and vortex for 5 seconds. What’s happening: solution C4 is a
high concentration salt solution. Since DNA binds tightly to silica
at high salt concentrations, this will adjust the DNA solution salt
concentration to allow binding of DNA, but not contaminants that
may still be present, to the spin filter.
15. Load approximately 675 ul of your DNA containing solution into a
spin filter tube and centrifuge at 10,000 x g for 1 minute at room
temperature. Remove the filter insert and discard flow through
from the collection tube – dump into a waste container or in the
sink. Replace the filter insert to its collection tube base. Add
another 675 ul of your DNA containing solution into the filter and
spin at 10,000 x g for 1 minute. Dump the flow through as before
and replace the spin filter into the collection tube base. Add the
remaining DNA containing solution to the filter and spin at 10,000
x g for 1 minute. Dump the flow through as above. Note: a total
of three loads for each sample processed are required. What’s
happening: the DNA is selectively bound to the silica membrane in
the spin filter device in the high salt solution. Contaminants pass
through the filter membrane leaving only DNA bound to the spin
filter.
16. Obtain solution C5 from your instructor. Add 500 ul of solution C5
to the spin filter and centrifuge at 10,000 x g for 30 seconds at
room temperature. What’s happening: solution C5 is an ethanol
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Urban Microbial Community Dynamics and Metagenomics
based wash solution used to further clean the DNA that is bound
to the silica filter. This wash removes residual salt and other
contaminants while allowing the DNA to stay bound to the silica
filter.
17. Discard the flow through from the collection tube as described
above.
18. Centrifuge the spin filter at 10,000 x g for 1 minute at room
temperature to remove any residual liquid from the filter or the
sides of the filter. What’s happening: this spin step removes the
residual solution C5 (ethnol wash). It is critical to remove all
traces of wash solution C5 because the ethanol can interfere with
many downstream DNA applications such as PCR, restriction
digests and gel electrophoresis.
19. Carefully place the dry spin filter in a clean, labeled, 2 ml
collection tube. Avoid getting any residual C5 solution on the filter
insert.
20. Obtain solution C6 from your instructor. Add 100 ul of solution
C6 to the center of the white filter membrane. Note: placing the
solution C6 in the center of the small white membrane will make
sure the entire membrane is wetted. This will result in more
efficient and complete release of the DNA from the silica filter
membrane. As solution C6 (elution buffer) passes through the
silica membrane, DNA that was bound in the presence of high salt
is selectively released by solution C6 (10 mM tris buffer) that lacks
salt.
21. Centrifuge at room temperature for 30 seconds at 10,000 x g.
22. Discard the spin filter. The DNA is in the collection tube is now
ready for downstream applications.
23. Store your DNA in the collection tube at -20 C.
g. Once you have eluted your final purified metagenomic DNA sample, quantify the
sample using the NanoDrop and/or by running your sample on a 1% agarose gel.
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Urban Microbial Community Dynamics and Metagenomics
Step 3
DNA quantification by NanoDrop
A detailed description for the use of the NanoDrop instrument is provided in the appendices. A
general description for the use of the NanoDrop is provided here:
1. In order to use the NanoDrop instrument you will need a P2 or P10 pipette and tips,
a small volume of elution buffer and control  (lambda) DNA of known concentration,
in addition to your PowerSoil DNA sample.
2. Your instructor will demonstrate the use of the NanoDrop instrument.
3. Add 1ul of sterile water to the pedestal, move the arm into place, and the open again
and use a kimwipe to gently wipe away the drop of water.
4. Place 1ul of the appropriate blank (the same solution you eluted your DNA with –
most likely C6 buffer from the PowerSoil kit) on the pedestal and close the arm.
Blank the instrument. Then open the arm and gently wipe away the blank with a
kimwipe.
5. Place 1 ul of a known concentration of  DNA control DNA or your sample on the
pedestal and close the arm. Type in the control or sample name into the sample
field on the computer. Now click on the “measure” icon to measure your sample.
After measuring, lift the arm and wipe away your sample.
6. Repeat step 5 with any additional samples.
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Urban Microbial Community Dynamics and Metagenomics
Step 4
PCR amplification of 16S rRNA gene from the metagenomic DNA sample.
Now that you have collected, isolated and quantified the genomic DNA from the microorganisms
picked up on your swabs from the environment, it is now necessary to amplify a specific region
of the bacterial 16S rRNA gene that will be used to identify what types of bacteria are present in
your sample (see below for more details on the structure of the 16 rRNA gene). Using universal
primers that recognize conserved regions of the bacterial 16S rRNA gene you will amplify a
variable region from the 16S rRNA gene. Because your metagenomic DNA samples contain
16S rRNA genes from all the different bacteria present in the sample, the universal primers will
amplify the targeted 16S rRNA gene region from all the different bacteria – and therefore the
PCR product will represent a mix of 16S rRNA regions. Sequencing the 16S regions later will
allow you to determine which bacteria were present in your sample as well as determining their
relative abundance.
Use the Qiagen HotStart kit (#203445). The detailed manufacturers protocol is included in the
appendices. The following list shows and example if the set-up for 10x 50 ul reactions:
Single reaction
Template
2 ul
Forward primer
2 ul
Reverse primer
2 ul
2X master mix
25 ul
dH2O
19 ul
Total
50 ul
10 (+1) reaction master mix
22 ul
275 ul
209 ul
506 ul
Notes: for inexperienced students who have not had practice pipetting, the instructor should
prepare the master mix. Students should not work directly with any of the stock reagents. It
may also be necessary to dilute the concentration of the metagenomic template DNA by 1:10 if
it above 10 ng/ul in concentration.
When preparing a master mix for 50 ul reaction volumes, add one additional reaction per 10
reactions to account for volume loss due to pipetting. For example, as shown above, if a master
mix is being made for 10 samples then the volumes should be calculated as for 11 (10+1)
reactions.
Prepare your template DNA for two PCR reactions. Pipette 18 ul of sterile, molecular grade
water into a clean microcentrifuge tube. Add 2 ul of your isolated metagenomic DNA sample to
the tube to make a 1:10 dilution of your template. Make sure your tube containing the 1:10
dilution is clearly labeled. You will set-up two PCR reactions with your DNA sample. One
reaction will use 2 ul of your undiluted sample as template for the PCR reaction and the second
reaction will use 2 ul of the 1:10 dilution of your DNA sample as template.
Your instructor will provide you the master mix and any other reagents you need. Each group in
the lab will be using a different “bar coded” forward primer so that samples can be combined
prior to sequencing later in the process. The bar code on your forward primer consists of a
stretch of 6-8 bases that are unique to this primer and can be identified by sequencing.
Because each group will use their own bar coded forward primer the primers cannot be added
to the master mix used by all groups in the lab. Your instructor will give you your bar coded
9
Urban Microbial Community Dynamics and Metagenomics
primer. Add the following components (in the order listed and check off each component after
you have added it) to each of your two PCR tubes:
1. Master mix (containing water, HotStart 2x mix and reverse primer): 46 ul
2. Forward primer: 2 ul
3. Template DNA*: 2 ul
*Remember that one of your PCR tubes will receive 2 ul of undiluted template DNA and the
other tube will receive 2 ul of the 1:10 dilution of the template DNA that you prepared above.
Your instructor will introduce you to the working of the thermal cycler. The thermal-cycler
should be set as follows (according to the HotStart basic protocol):
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Initial heat activation
Denaturation/melting
Annealing
Extension
Cycle to step 2 for 34 times
Final extension
Hold/Pause
15 min
30 sec
45 sec
45 sec
95C
95C
55C
72C
10 min
Indefinitely
72C
10C
On the graphical display you can watch the how the instruments runs through the cycles and an
estimate of when your samples will have finished their run. After the PCR run make sure your
instructor has your samples and that they have been places in a box or rack for storage at -20C.
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Urban Microbial Community Dynamics and Metagenomics
Step 5
Analysis of PCR reactions by agarose gel electrophoresis
In order to check whether or not a PCR product of the expected size was amplified you will run
a fraction of your PCR reactions on a 1% agarose gel to visualize the DNA bands that are
present in your two PCR reactions.
You will prepare a 1% agarose gel as follows:
1. Make sure you have gloves and safety glasses on.
2. In a ~250 ml Erlenmeyer flask measure out 100 ml of 1X TAE buffer.
3. Weigh out 1 gram of agarose powder and add it to the Erlenmeyer flask with the
buffer.
4. Place Saran-Wrap over the mouth of the flask and then place the flask on the
balance and zero the balance.
5. Carefully microwave the agarose in the buffer at ~70% power in a microwave for
2-4 minutes. Do this in 30 second increments to avoid having the liquid boil over.
All the agarose powder should go into solution and the buffer should be clear
with now powder of crystalline agarose floating around.
6. Allow the melted agarose to cool for 5-8 minutes. Then carefully add 2 drops of
ethidium bromide (this is a carcinogen, so make sure you are wearing gloves and
that the instructor is present to assist you). Carefully swirl to mix.
7. Pour the melted agarose into the casting cassette and insert the appropriate
comb to create the wells.
8. Let the agarose cool and gel for ~30 minutes.
Take two clean microcentrifuge tubes and label them to correspond to your two PCR samples.
Carefully transfer 5 ul of each PCR reaction into its own microcentrifuge tube. Now add 2 ul of
the orange 6x gel-loading buffer to each of your two samples. Flick the tubes to mix your
sample with the loading buffer. Your instructor will demonstrate how to load the gel. Add all 7
ul of your sample plus loading buffer into a lane on the gel. Make sure you make note of the
lanes your samples are in. Your instructor will also make sure that a size standard is also
loaded in one or more lanes of the gel.
The gel should be run at ~80-100 volts for 45-75 minutes. Make sure not to run the gel so long
that the dye front runs off the gel or at such a high voltage that the gel box heats up. When the
gel has finished running your instructor will show you how to visualize the gel on a UV light box
and how to capture images of the gel on the camera.
Compare the size of any bands in your sample lanes with the size standard and record your
results.
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Urban Microbial Community Dynamics and Metagenomics
Step 6
PCR clean-up and submission of metagenomic 16S amplicon for sequencing
Before the final quantification of your 16S rRNA gene amplicon and subsequent submission for
next generation sequencing, the other components of the PCR reaction mix (unincorporated
nucleotides and primers as well as polymerase enzyme) must be removed from your sample. A
silica-based column purification system is used to bind your amplicon DNA while washing away
the other components, and then finally eluting the amplicon DNA from the column in water or
buffer. You will use the Qiagen MiniElute PCR purification kit to do this (cat# 28004). You
should only continue to this clean-up step if the gel analysis of your PCR reactions showed a
band of the expected size. Do not try to clean-up your PCR reaction if there is clearly no band
present or if the band present is not the correct size or if there are multiple bands present (not
including any faint low molecular band resulting from the presence of unincorportated primers).
Your instructor will demonstrate the use of the MiniElute kit. A detailed protocol from the
manufacturer is provided in the appendices. The basic steps for purification of the amplicon
DNA are as follows:
1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. For
example, add 250 μl of Buffer PB to 50 μl PCR reaction.
2. If pH indicator I has been added to Buffer PB, check that the color of the mixture
is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium
acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
4. To bind DNA, apply the ~25 ul sample to the MinElute column and centrifuge for
1 min at 10K x g.
5. Discard flow-through. Place the MinElute column back into the same tube.
6. To wash, add 750 μl Buffer PE to the MinElute column and centrifuge at 10K x g
for 1 min.
7. Discard flow-through and place the MinElute column back in the same tube.
Centrifuge the column for an additional 1 min at maximum speed. NOTE:
Residual ethanol from Buffer PE will not be completely removed unless the flowthrough is discarded before this additional centrifugation.
8. Place the MinElute column in a clean 1.5 ml microcentrifuge tube. To elute DNA,
add 10 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the
membrane, let the column stand for 1 min, and then centrifuge for 1 min. Note:
Ensure that the elution buffer is dispensed directly onto the center of the
membrane for complete elution of bound DNA. The average eluate volume is 9 μl
from 10 μl elution buffer volume.
9. Use than NanoDrop instrument (as described above) to quantify 1 ul of your
eluate containing the PCR amplicon.
10. Record the concentration and purity ratios of your PCR amplicons.
Your instructor will collect your samples and coordinate the shipping of the samples to the
commercial sequencing facility.
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Urban Microbial Community Dynamics and Metagenomics
1.
Optional:
h. Dilute a portion of your sample by adding 100 ul into 900 ul of media in a sterile
microcentrifuge tube
i. Plating
i. Plate 100 ul of your undiluted sample on an NA plate
ii. Plate 100 ul of your undiluted sample on an MSA plate
iii. Plate 100 ul of your 1:10 dilution on an NA plate
iv. Plate 100 ul of your 1:10 dilution on an MSA plate
j. Seal your plates in Parafilm and place them in the container specified by your
instructor. The plates will be incubated at room temperature.
13