Bioinformatic Analysis of Microbial Diversity:
Isolation, Amplification, Cloning and Sequence Analysis of 16S rRNA
Sequences from Natural Microbial Communities
Bioinformatic analysis of nucleotide sequences of the small ribosomal subunit genes (16S
and 18S rRNA genes) has become the method of choice to identify the microbes or microbial
genera present in natural communities. These sequences are easily obtained from metagenomic
DNA by amplification and cloning of 16S and 18S genes. Because approximately 99% of
naturally occurring microbes cannot be cultured in the laboratory, bioinformatic analysis is the
sole means of identification. An approximate timeline for these experiments is:
Day 1: Isolate metagenomic DNA from soil sample (0.5 hr), amplify 16S sequences by PCR (2.5 – 3
hr); ligate PCR products to the vector pCR2.1 (1 hr); transform competent DH5 cells
with the ligation mixture (1.5 - 2 hr); and plate transformation mix to selective media to
identify plasmid-bearing cells (0.5 hr)
Day 2: Inoculate cultures of transformants (0.5 hr)
Day 3: Isolate plasmid DNA containing 16S genes from these cultures ( 1 – 2 hr)
Day 4: Subject DNA to nucleotide sequence determination (24 hr)
Day 5: Bioinformatic analyses of 16S nucleotide sequences
1
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The secondary structure of the 16S ribosomal RNA molecule, showing its single-stranded and base-paired regions.
The 16S rRNA interacts with 21 proteins present in the 30S small ribosomal subunit through the numerous stemloop structures shown in the diagram above. (Cover of Science 309, Sept., 2005.)
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Developed by Ann Sliski and Karen Malatesta
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Laboratory 1: Isolation of Metagenomic DNA from Soil and
Amplification of 16S rRNA Genes
Objectives of Laboratory 1A:
1. Isolate metagenomic DNA from a soil or sample
2. Subject eight reactions specific for the 16S gene of a microbial domain or genus to amplification
by PCR
3. Prepare competent DH5 cells
Flow Chart of Laboratory 1A:
Isolate Metagenomic
DNA from Soil
Set up Eight PCR Reactions
Specific for 16S Genes
Prepare Competent
DH5 Cells
“It’s just astounding to see how constant, how conserved, certain sequence motifs—proteins, genes—have been over
enormous expanses of time. You can see sequence patterns that have persisted probably for over three billion years.
That’s far longer than mountain ranges last, than continents retain their shape.”
Carl Woese, 1997, In Perry and Staley, Microbiology.
INTRODUCTION: From the late 1800’s, when Koch cultured the anthrax bacillus and
proved it was the causative agent of anthrax, until the mid-1980’s, scientists were confident they
had identified most microbes present in the biosphere, estimated to include 107 to 109 different
species of bacteria (Schloss and Handelsman, 2004). However, this identification was absolutely
contingent on the ability to these microbes in the laboratory. Therefore, when new evidence
gathered from aquatic and terrestrial ecosystems indicated that more than 99% of the
microorganisms present in the environment could not be cultured in the laboratory and, thus,
could be identified only by molecular means, shockwaves shook the scientific community to its
core (Amann et al., 1995).
In 1977, Woese and Fox had proposed using ribosomal RNA (rRNA) gene sequences to
classify bacteria and eukaryotes (Woese and Fox, 1977). The genes encoding the small
ribosomal subunit (SSU, the 16S rRNA gene in bacteria and the 18S gene in eukaryotes; see
Figure on next page) were selected because all species encode homologues of these genes and
also because both the 16S and 18S genes contain both conserved and variable regions. The
application of this method, known as ribotyping, resulted in a re-classification of organisms into
three kingdoms (Bacteria, Archaea, and Eucaryotes) rather than the five kingdoms that had been
previously recognized. The advent of the Polymerase Chain Reaction greatly facilitated
ribotyping, and The Ribosomal Database Project (Cole et al., 2005; http://rdp.cme.msu.edu/) was
established for 16S and 18S sequences and is now in its second generation. RDP II curates over
101,600 16S rRNA gene sequences and includes both sequences amplified directly from the
environment without prior culturing as well as sequences obtained from cultured microbes.
Ribotyping has led to an enormous increase in the number of bacterial phyla, currently about 52,
half of which are composed only of uncultured bacteria (Schloss and Handelsman, 2004; Rappe
and Giovannoni, 2003). In fact, in July 2005, the number of 16S sequences from environmental
organisms surpassed that from cultured organisms.
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One method of studying uncultured microbes is to analyze genomic DNA isolated from a
community of organisms. This type of study, in which DNA is obtained directly from the
environment without prior culture, has spawned a new field known as metagenomics (Rondon et
al. 2000; Handelsman. 2004). Metagenomics has not only facilitated analyses of genomic
complexity and evolution but also resulted in the isolation of novel clones that express many
different enzymatic activities, including anti-microbial compounds.
Today, you will first isolate metagenomic DNA from your soil sample and use this DNA to
set up PCR reactions using primers specific for the 16S rRNA genes of either Bacteria or
Archaea. Your reactions will be amplified over the noon hour, after which you will ligate the
products of one PCR reaction to DNA of the vector pCR2.1. Later today, you will complete the
process of cloning the amplified 16S rRNA genes from your soil sample by transforming the
ligated products into E. coli. This procedure produces clones, which are exact copies.
The polymerase chain reaction (PCR) has revolutionized not only molecular biology but
also numerous other scientific fields. PCR is a method by which a defined region of DNA is
synthesized from minute amounts, even as little as a single DNA molecule, to yield quantities of
DNA sufficient for detailed studies and analysis. This technique has become widely used in
genetic diagnosis and forensics, as well as in innumerable basic research applications. The
requirements for PCR include: a DNA polymerase to synthesize DNA, a DNA template for the
polymerase to copy, the four deoxynucleoside triphosphates (dATP, dGTP, dCTP and dTTP)
that are the building blocks of DNA, short DNA molecules (oligonucleotides) to serve as
starting points or primers for DNA synthesis, and suitable reaction conditions for the DNA
polymerase to synthesize DNA. PCR is usually performed using a thermally stable DNA
polymerase known as Taq polymerase, which was isolated from Thermus aquaticus, a
thermophilic bacterium that inhabits hot springs in Yellowstone National Park. In the reactions
you will set up today, the template will be the DNA you isolated this morning from the microbes
in your soil sample. The primers are short (15-25 bp) DNA molecules that function as starting
sites for Taq polymerase to begin synthesizing DNA and are specific for the chromosomal region
being amplified, in this case the 16S rRNA genes of soil microbes. The sequences of the primers
are very important: they must be the exact complement (A pairing with T and G pairing with C)
of sequences flanking the chromosomal region to be amplified.
The basic PCR cycle is composed of three steps or reactions, each of which is performed at a
different temperature. In the first step, the template DNA is denatured at high temperature for a
short time (94o C for 1 min in our reactions). In the second step, the temperature is lowered to
allow the primers to anneal to the template DNA, again for a short time (20 sec at 43o C followed
by 30 sec at 58o C). The 43o C incubation is necessary because some primers have low melting
temperatures. In the third step, the temperature is raised to the optimal temperature for the DNA
polymerase to synthesize DNA (72o C for 1 min). These steps are diagrammed in the Figure on
the next page. Although the procedure is very rapid compared to many other techniques (a single
three-reaction cycle usually requires less than four minutes), it is necessary to repeat this cycle
thirty times to synthesize enough DNA for you to clone and also analyze by agarose gel
electrophoresis.
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In addition to being thermostable, Taq has the unusual characteristic of adding an extra “A”
to the 3’ end of each sequence it amplifies. This additional base is very useful in the process of
ligating the PCR products Taq produces to a plasmid vector. Without this additional “A”, the
PCR products would have blunt ends, which ligate poorly. Consequently, far fewer ligation
products will be produced. Plasmid vectors like pCR2.1 were created for the purpose of cloning
PCR products by addition of a “T” to each of its 5’ ends to make these ends complementary to th
e3’ ends of the PCR products.
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II. EXPERIMENTAL PROCEDURES: Wear gloves and work only with your sample
to avoid contaminating it with other microbes. Use cotton-plugged aerosol resistant tips
(ARTips) at all times.

A. Processing Your Sample of Soil:
1. Obtain a new Ziploc plastic bag and dump your soil sample  Label this bag with your initials
and the date.
from the tube you used to collect it into the bag.
2. Composit (mix) your soil sample by inverting and  Don’t open the bag so the soil
will remain sterile.
massaging the bag several times.
3. Although you will not sieve your soil sample today, 
sieving removes large particles and also helps mix the soil.
B. Isolation of Metagenomic DNA from Your Soil Sample:  You will use the Powersoil DNA
Isolation Kit from MO BIO
This procedure was delineated by MO BIO Laboratories, Inc.,
Laboratories, Inc. (#12888-50 or
and is more rapid than comparable DNA isolation kits.
12888-100)
1. Obtain an Isotherm and ice from near the large sink before  Spatulas can be sterilized by
rinsing
them
in
alcohol.
beginning. Be sure to wear gloves and ask for help weighing
Weighing instructions are next to
your sample if needed.
the balances.
2. Use a sterile spatula and a small weigh boat to weigh out  0.25 g of damp soil is about the
size of a large pea. Put the
250 mg (0.25 g) of soil.
remainder of your soil into the
cold box until tomorrow.
3. Add this 250 mg soil to a PowerBead Tube labeled with  The PowerBead tube contains
beads and a buffer to help
your initials. This tube contains small beads, which
disperse soil particles, dissolve
physically break cells open during the vortexing step.
humic acids and protect against
DNA degradation. Humic acids
can inhibit a variety of chemical
reactions, including PCR.
4. Vortex gently to mix and disperse the soil.
5. Check that Solution C1 does not contain a precipitate.
 If a precipitate is opresent, heat
this solution to 60 C until the
precipitate is dissolved.
6. Add 60 l of solution C1 to the tube and invert several times  Solution C1 contains the
detergent SDS and other agents
to mix.
to completely lyse cells. SDS is
an anionic detergent that disrupts
lipids and fatty acids in cell
membranes.
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7. Secure your PowerBead Tube horizontally to a vortex mixer  If tape is used, check the
apparatus often because tape can
using the MO BIO vortex adapter tube holder or using tape
easily become loose.
to fasten it to a flat-bed vortex pad.
 This step is critical for complete
8. Vortex at maximal speed for 10 minutes.
lysis of the cells, which is caused
by the chemical reagents in the
PowerBead Tube as well as
mechanical collisions between the
beads and cells.
9. Place your PowerBead Tube into a microfuge.
 Make sure that the tube rotates
freely in the microfuge without
rubbing.
10. Centrifuge your tube at 10,000 x g for 30 sec at room  Do not exceed 10,000 x g or the
tube may break.
temperature. (Conversion charts at Eppendorf URL)
11. Use your P200 to transfer the supernatant to a clean 2-ml  You should have 400 – 500 l
supernatant, but the exact volume
collection tube that you have labeled with your initials.
and color of the supernatant is
unimportant.
12. Add 250 l of Solution C2 and vortex for 5 sec.
 Solution C2 will precipitate
13. Incubate at 4o C for 5 min.

organic and non-organic material,
including cell debris and protein.
14. Centrifuge your tube at rt (room temperature) for 1 min at 
10,000 x g.
15. Avoiding the pellet, use your P200 to transfer up to 600 l  This is easy if you keep the pipet
tip just below the meniscus of the
of supernatant to a clean 2-ml collection tube.
supernatant.
16. Add 200 l of solution C3 to your tube and vortex briefly.
 This solution also precipitates
17. Incubate at 4o C for 5 min.

18. Again centrifuge your tube at rt for 1 min at 10,000 x g.

organic and inorganic substances.
19. Again, avoid the pellet and use your P200 to transfer up to 
750 l of supernatant to another clean 2-ml collection tube.
20. Add 1.2 ml of Solution C4 to the supernatant, being careful  C4 contains a high concentration
of salt, which will ensure that
that the solution doesn’t overflow the rim of the tube.
DNA binds tightly to the silica
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spin filters.
21. Mix very well by vortexing 5 sec and inverting tube several 
times.
22. Load approximately 675 l onto a spin filter and centrifuge 
at 10,000 x g for 1 min at rt.
23. Discard the flow through into a waste tube or beaker.

24. Add an additional 675 l supernatant onto the spin filter.

25. Centrifuge this tube at 10,000 x g for 1 min at rt again.

26. Again discard the flow through into the waste tube and  Three loads of supernatant are
required.
load the remaining supernatant onto the spin filter.
27. Spin again at 10,000 x g for 1 min at rt and discard the flow  The DNA in your sample is now
bound to the silica membrane in
through into the waste tube again.
the spin filter.
28. Add 500 l of Solution C5 and centrifuge at rt for 30 sec at  Solution C5 contains ethanol to
wash contaminants from the
10,000 x g.
precipitated DNA on the silica
filter.
29. Discard the flow though from the 2 ml collection tube.

30. Centrifuge the spin filter at rt for 1 min at 10,000 x g.
 This
spin
solution C5.
removes
residual
31. Carefully place your spin filter into a clean 2 ml collection  Take care not to splash any
solution C5 onto the spin filter.
tube.
32. Add 100 l of Solution C6 to the center of the white filter  Solution C6 (10 mM Tris buffer)
will elute the DNA from the spin
membrane.
filter. Placing this solution on the
center of the filter will ensure that
all areas are wetted.
33. Centrifuge at rt for 30 sec at 10,000 x g.

34. Discard the spin filter.

35. The metagenomic DNA you have just isolated is now ready 
for amplification by PCR as described next or for other
applications.
The DNA should be kept on ice
or stored frozen (-20o to -80o C)
until ready for use.
You will now use this DNA to set up eight PCR reactions that  Some reactions will amplify a
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Princeton University
genus or species-specific 16S
rDNA, and others a universal
rDNA.
are specific for the 16S rRNA gene.
One partner should follow the directions in Section C below to 
make an E. coli control for PCR, while the second partner
should harvest DH5 cells as described in Section D below.
C. Making a Positive E. coli Control for PCR (Partner #1):

1. Obtain a clean 1.5-ml screw cap tube and add 50 l sterile  Label this tube with your initials
and “K12” to denote E. coli K12.
water (clear tube, blue dot) to it.
Keep the tube of water.
2. Obtain the stock plate of DH5 you worked with yesterday.
 DH5 is an E. coli K-12 strain.
3. Light your Bunsen burner with the striker.

4. Flame your loop until it glows red and touch it to the agar at  This cools the loop.
the side of the plate away from any colonies.
5. Use your loop to pick up a colony that is small to medium in 
size.
6. Transfer some E. coli cells to the water by moving the loop 
rapidly through the water in your tube.
7. Screw the cap onto your tube tightly.

8. Boil the water and E. coli for 5 min in a heating block.
 This lyses the cells, releasing
their genomic DNA.
9. When the boiling step ends, put your tube in ice until you  Preparing a sample like this
control would be very easy to do
are ready to use it to set up a PCR reaction specific for E.
in your classrooms.
coli.
D. Preparation of Competent DH5 Cells (Other Partner):  The growth on this plate should
be dense because the loop was
Begin preparing competent DH5 following the instructions
not flamed after making the initial
below. Be sure to use the plate that has been grown 5 days.
streak, and the streak made a tight
zigzag pattern.
1. Obtain an LB plate onto which DH5 was streaked five days
ago from the front bench as well as an orange-capped tube of
LB broth.
 This plate was incubated at room
temperature for 5 days and
should contain rather dense
growth.
2. Use your P1000 to add 1 ml LB broth from the orange-  Set your P1000 to “1-0-0”.
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capped tube to the surface of the plate and roll the plate to
distribute the broth over as much of the surface as possible.
3. Locate the glass spreader (a glass rod bent into an “L” shape)  Place your burner in a spot
where neither you nor your
at your bench and light your Bunsen burner with the striker.
partner has to reach over it.
4. Sterilize your spreader by dipping it into the jar of ethanol at 
your bench and setting it aflame by putting it briefly into the
flame of the burner.
5. The flame on the spreader will burn for only a couple of 
seconds before going out.
6. Lift the lid off the plate of DH5 and hold the lid above the 
plate to prevent contamination.
7. Briefly touch the spreader to an area of the plate that does not  This cools the spreader.
have any bacterial growth.
8. Move the spreader carefully across the entire surface of the 
plate to resuspend as many DH5 cells as possible in the L
broth.
9. Tilt the plate at an angle by leaning it against a test tube rack 
to allow the broth to collect at the lowest point. Use your
spreader to sweep broth into the puddle at the lowest point.
10. Use a sterile transfer pipet to transfer the resuspended cells  Label this tube with your initials.
to a 1.5 ml microtube.
11. Lay the plate flat on the bench and rinse the spreader with 
another 0.5 ml of LB while holding the spreader over the
plate.
12. Move the spreader over the surface of the plate again to 
resuspend any remaining cells in the broth.
13. Again lean the plate against a rack and allow the broth to  Again use the spreader to sweep
liquid into the puddle.
collect at the lowest point.
14. Use another sterile transfer pipet to transfer this second  Flick this tube to mix the
contents.
aliquot of resuspended cells to the same 1.5 ml tube.
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15. Let the cells sit undisturbed at rt for exactly 2 hr 15 min  This relatively long incubation
produces more competent cells.
over lunch.
16. Before proceeding, turn off your burner and wipe down 
your bench with 70% ethanol from a wash bottle. Continue
to prepare cells after lunch.
E. Setting up PCR Reactions for 16S rRNA Genes: Each lab
pair will cooperate to set up seven PCR reactions specific for
the 16S rRNA genes of different microbes using the DNA you
and your partner just isolated from soil. The eighth reaction will
be a positive control using the E. coli DNA you just made. Both
partners should cooperate to make these eight PCR reactions.





Continue to use ARTips when
pipetting in order to prevent
contamination. Additional racks
are available if needed.
Please change your gloves now
before beginning.
1. The first partner should take an Isotherm and ice to obtain  The primer mixtures contain
equal parts of forward and reverse
seven tubes containing the following primer mixtures from
primers.
the front bench.
a. Universal primers for bacteria (0.65 ml tube labeled “FR”)
 Biological Procedures Online.
www.biologicalprocedures.com/b
po/arts/1/6/m6.htm
b. Universal primers for archaea (0.65 ml tube labeled “A1”)
 Baker et al. 2003. J. Microbiol.
Meth. 55: 541.
c. Primers for Pseudomonas (0.65 ml tube labeled “Pseu”)
 Milling et al. 2004. Plant & Soil
266: 23.
d. Primers for fungi, protists, and green algae (0.65 ml tube labeled  White et al. In PCR Protocols, A
Guide
to
Methods
and
“NS”)
Applications. P. 315.
Kuske et al. 1998. Appl. Environ.
Microbiol. 64: 2463.
e. Primers for high G+C gram + bacteria, which includes Actinomyces
(0.65 ml tube labeled “Act”)

f. Primers for Bacilli (0.65 ml tube labeled “Bac”)
 Kuske et al. 1998. Appl. Environ.
Microbiol. 64: 2463.
Biotechnologies
g. Primers for E. coli K12 (0.65 ml tube labeled “E”). You will  Epicentre
(www.epibio.com)
use these primers to set up two PCR reactions.
2. Obtain eight clear 0.5 ml Ready-To-Go PCR Bead Tubes  Each Ready-To-Go PCR Bead
contains Taq polymerase, the four
from the front bench.
3. Tap each tube gently on your bench to ensure the bead is at
the bottom of the tube before opening that tube.
dNTP's, MgCl2, KCl2, and TrisHCl buffer.
4. Label the tops and sides of seven of these tubes using a fine-  Label the eighth tube “E-C” for
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tipped black marker with one of the seven letters/acronyms
to designate the different primers, e.g., “A1”, “FR”, etc.
“E. coli control”.
5. Using your P20 set to “2-0-0” with a fresh 20 l ARTip, add  For example, add 20 l “A1”
primers to the tube labeled “A1”.
20 l of each of the primer mixtures to the appropriately
Each mixture contains 0.5 l each
labeled Ready-To-Go Bead Tubes and the tube labeled “Eof forward and reverse primers
C”. Be sure to use a clean tip for each primer mixture.
(10 M) and 19 l H2O.
6. Flick all eight tubes gently but well with your fingers until  Keep these Ready-To-Go tubes
cold in ice as often as possible.
the PCR beads are thoroughly dissolved.

7. Move the tube labeled “E-C” away from the other seven  You will add a different DNA
template to the “E-C” tube.
tubes.
8. Using your P20 with a fresh 20 l ARTip, transfer 5.0 l of  Do not add soil DNA to the tube
labeled “E-C”.
your soil DNA to each of the seven Ready-To-Go PCR
Bead tubes you just prepared.
9. Flick these tubes gently but thoroughly to mix.

10. Transfer 5.0 l of the E. coli DNA you prepared earlier to 
the tube labeled “E-C”.
11. Flick the “E-C” tube to mix the contents and spin all eight  This brings all droplets to the
bottom of each tube.
tubes 10 sec in the microfuge.
12. One partner should obtain one strip of eight 0.2 ml PCR  Be sure to wear gloves when
obtaining these tubes.
tubes and a PCR rack from the front bench. (Omit steps 1214 if your cycler accommodates 0.5 ml tubes.)
13. Use a black marker to label each PCR tube with at least  Label the top and hinge of each
tube if possible.
one of your initials and one of the seven acronyms (and
another “E-C”) denoting the different primer pairs.
14. Set your P200 to “0-3-5” (the bead adds volume) and transfer  Transfer the contents of the “EC” tube last.
the entire contents of each Ready-To-Go Bead tube into the
PCR tube with the matching label.
15. Return the tube containing the soil DNA you isolated to the  Make sure this tube is clearly
marked with your initials and
front of the lab for storage.
“Soil DNA”.
Bring your strip of eight PCR tubes to the front of the lab  Your tubes will be put into an
automated thermal cycler that
and put them into the PCR rack.
has been programmed for the
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following conditions.
94 C 3 min

30 cycles:
Denaturing time and temperature: 1 min at 94o C


o
Annealing time and temperature: 20 sec at 43o C followed  Annealing at 43o C is necessary
because some primers have low
by 30 sec at 58o C
melting temperatures.
Extending time and temperature: 1 min at 72o C
72o C 10 min


Laboratory: Analysis and Cloning of 16S PCR Products
Objectives of Laboratory 1B:
1.Set up a ligation reaction using your “FR” PCR products and the vector pCR2.1
2. Analyze your PCR products using e-gel electrophoresis
3. Finish preparing competent DH5 cells
4. Transform your ligation reaction into these DH5 cells
5. Plate transformants onto selective plates
Flow Chart of Laboratory 1B:
Set up a
Ligation
Analyze PCR Products
using Electrophoresis
Transform DH5
with Ligation Mix
Plate to Select
Transformants
I. INTRODUCTION: This morning you isolated metagenomic DNA from soil and set up
several PCR reactions to amplify 16S sequences from this DNA. You will now continue your
experiment by setting up a ligation reaction to ligate or tie the PCR products in your “FR”
reaction to the plasmid vector, pCR2.1. This process is called cloning because clones are exact
copies. Cloning has three important components or ingredients: 1) fragments of DNA to be
cloned (your PCR products in today’s experiment); 2) a vector (an engineered plasmid or virus)
into which the cloned fragments are inserted (pCR2.1 in your experiment); and 3) the enzyme
DNA ligase that forms phosphodiester bonds between the DNA fragments and the vector DNA.
The vector pCR2.1 has several features that are essential for cloning: a replication origin
which enables it to replicate independently of the host bacterial chromosome, genes for
resistance to ampicillin (Ampr) and kanamycin (Kanr) which are used to select for cells that
contain pCR2.1, a region known as a polylinker which contains cloning sites for 14 restriction
enzymes, and a short segment (the LacZ  fragment), which contains the regulatory sequences
and coding information for 146 amino acids of the E. coli -galactosidase (LacZ) gene. galactosidase is an enzyme that breaks down lactose into glucose and galactose. The polypeptide
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encoded by this region of LacZ is known as the  subunit of -galactosidase and is the basis for
an easy assay that will allow you to determine visually whether a foreign DNA fragment has
been inserted into the polylinker of pCR2.1. A diagram of pCR2.1 is shown on the next page.
In addition to being thermostable and able to remain active despite the elevated temperatures
used for PCR, Taq has another advantage when used for PCR. Specifically, Taq has the unusual
characteristic of adding an extra “A” to the 3’ ends of each DNA sequence it synthesizes. These
additional bases are very useful in ligating PCR products to a vector because without these
additional “A’s”, far fewer ligation products would be produced. Plasmid vectors like pCR2.1
that are used for cloning PCR products have been modified by addition of “T’s” to make the 3’
ends of the vector molecules complementary to the ends of the PCR products.
After you set up your ligation reaction, you will analyze all your PCR products using agarose
gel electrophoresis. Then you will transform your ligation mix into competent DH5 cells you
will prepare. Later today, you will plate your transformation mix onto special plates to select for
cells that have taken up a plasmid.
T7 priming
site
14
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
The diagram of pCR2.1 above illustrates several features of the vector that are essential in cloning,
including 14 sites for restriction enzyme cleavage that flank the cloning site into which an exogenous
DNA fragment can be inserted.
15
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
II. EXPERIMENTAL PROCEDURES: One partner should continue preparing
competent DH5 cells (following the instructions in Section A below) while the second partner
sets up a ligation reaction (as directed in Section B below):
A. Partner #1- Preparation of Competent DH5 Cells:

1. Obtain fresh ice in your Isotherm and collect small tubes  Keep these tubes on ice. Calcium
ions make bacterial cells permeable
containing sterile solutions of 10, 30 and 100 mM CaCl2
so the cells take up DNA.
from the front bench.
2. When the DH5 cells you harvested earlier have incubated  Pour the LB broth supernatant
carefully into the waste container on
at rt for 2 hr and 15 min, spin the cells at 5 K rpm for 1min
your bench.
and pour off the supe carefully.
3. Add 1.0 ml 10 mM CaCl2 to the cells and flick the tube to  Cells become quite fragile when
treated with CaCl2, so never vortex
mix the contents well.
these cells.
4. Spin cells at 5 K rpm for 1 min and carefully pour off the  Watch the pellet as you pour and
discard the supe into the waste
supernatant.
container on your bench.
5. Add 0.3 ml of cold 30 mM CaCl2 to the cells and flick the  If the pellet does not resuspend well,
use your P200 set to 1-5-0 and a tip
tube to mix the contents well.
to squirt CaCl2 at the pellet to
resuspend the cells.
6. Leave these cells on ice for 10 – 20 min.

7. Spin cells at 5 K rpm for1 min again and pour off supe.
 Watch the pellet as you pour.
8. Flick the tube with your fingers to partially resuspend the  If the pellet is difficult to resuspend,
use your P200 again to resuspend
cells.
the cells.
9. Add 0.3 ml cold 100 mM CaCl2 and use your P200 to  Adjust the volume of 100 mM CaCl2
depending on the number of
resuspend the cells thoroughly.
transformations planned. Only 0.1
ml cells is needed for each
transformation.
10. Leave these cells on ice for 1 – 2 hr until your ligation 
reaction is complete.
B. Partner #2 -Ligation of Your PCR Products to pCR2.1:  Ligation reactions involving PCR
products must be carried out within
You will now set up one ligation reaction between the PCR
24 hrs of the completion of PCR, so
products in your “FR” PCR tube and purified DNA of the
the extra “A’s” added to the PCR
vector pCR2.1.
products by Taq will not be cleaved
off.
16
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
1. Put on a fresh pair of gloves and obtain another 
Isotherm and some ice.
2. Take your Isotherm to retrieve the strip of eight tubes  Keep these tubes on ice at all times.
containing your PCR products from the front table.
3. Use your “FR” PCR products to set up a ligation mixture  DNA ligase, which is purified from
bacteriophage T4, joins DNA
containing the following components (set the other
molecules together by forming
seven tubes of PCR products aside for now) in the order
phosphodiester
bonds
between
listed below:
them.
5.25 l sterile H2O
0.75 l “FR” PCR products (with “A’s” at 3’ ends)
1.00 l 10X ligase buffer (purple tube with black dot)
2.00 l pCR2.1 DNA (with “T’s” at 3’ ends)
9.00 l total volume
 Clear tube, blue dot


 Green 0.65 ml tube

4. Close the cap of the microtube, flick the tube vigorously 
to mix and spin it briefly in the microfuge.
5. Add 1 l T4 DNA ligase (purple tube) to this tube, flick it 
to mix and pulse spin the tube again.
6. You will now let this reaction incubate for 1 - 2 hr on  Be sure to record the time that this
incubation begins.
your bench at room temperature. Two hours is preferred.
C. Preparation of Selective Plates: Each lab pair should  Ampicillin and kanamycin are
antibiotics used to select Amp or
obtain three LB + Amp + IPTG + X-Gal plates (one black
stripe, one green stripe, one light blue stripe and one dark blue
stripe) from the front bench.
Kan resistant cells, X-Gal is an
artificial
substrate
for
galactosidase and IPTG (isopropyl-thiogalactopyranoside)
is
an
inducer of the Lactose operon.
1. Label the bottoms of these plates with your initials and 
the date.
Use a black marker for labeling.
2. Label two plates with “Lig”, your initials and the date.

3. Label the third plate “no DNA”, your initials and the date.

D. Preparing Your PCR Products for Electrophoresis:

1. Add 2.0 l loading dye (clear tube, purple dot) to each of  Do not discard this tube of loading
17
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
dye.
your eight PCR tubes.
2. Flick the tubes with your fingers to mix in the dye and  This pulse spin will bring the
sample to the bottom of the tube.
spin these tubes in the microfuge for a few seconds using
black adaptors.
E. Preparing your E-Gel for Electrophoresis: You and
your partner will use an e-gel for electrophoresis because
they are relatively easy to use and run rapidly. E-gels
contain ethidium bromide, which intercalates into DNA and
fluoresces under ultraviolet illumination. Prepare your gel as
described on the next page. Directions will be next to your
e-gel base and also reviewed by Dr. Malatesta or Dr. Sliski
before you begin.





1. Use scissors to open the package containing the e-gel.

Ethidium bromide is a mild
carcinogen, so you should always
wear gloves when working with egels.
2. For red bases, plug the E-gel Power base into an electrical  Older black e-gel bases may also be
used. Consult the manual for proper
outlet using the adaptor plug.
use.
3. With the comb in place, insert the gel into the apparatus, 
inserting the right edge first.
4. Press firmly at the top and bottom to seat the gel in the  When the gel is seated correctly,
you will hear a snap and a steady
base.
red light will illuminate.
5. The Invitrogen logo should be located at the bottom of the 
base, close to the positive pole, as shown below.
6. It is necessary to pre-run the e-gel for 2 minutes prior to 
loading your samples.
a. For red bases, press and hold either button on the Power  The flashing light indicates that
the 2–minute pre-run has started.
18
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University


1) When the pre-run ends, the current will automatically 
shut off, the flashing green light will change to a 
flashing red light, and the Power Base will beep.

 The light will change from a
2) Wearing gloves, press and release either button on the
 flashing red to a steady red.
Power Base to stop the beeping.
Base until the red light turns to a flashing green.
b. For black bases, pre-run the gel with the comb in place 
for 1 – 2 min at 60 – 70 V. Then turn off the power
supply.
8. Use both hands to remove the comb by gently lifting it and  Do this for both red and black
bases.
rolling it slowly toward you.
9. Obtain one tube of the 123 Base Pair Ladder (yellow tube)  The 123 Base Pair Ladder is a
mixture of fragments that differ in
from the front bench.
size by 123 base pairs.
10. Load 20 l of the 123 bp Ladder (yellow tube) into lane 1 of 
your e-gel.
11. Load 20 µl of each soil PCR product sample into one well  Be sure to record the order in
which your samples are loaded.
in the order in which the primers are listed on p. 34.
12. Be careful not to introduce bubbles while loading, as they  You can avoid introducing bubbles
into your sample by setting your
will cause bands to distort.
Pipetman to 20 l, which is the
exact volume you want to load.
13. Add 20 l H20 (clear tube, blue dot) to any empty wells.

14. For red bases, press the 30 min button to begin the run.
 Check that the dye is moving out
of the well.
15. For black bases, run your gel at 60 to 70 volts for  Do not run longer than 60 minutes
because longer run times will
approximately 30 to 40 minutes until the blue dye touches
damage the gel.
the black label at the bottom of the gel. Turn off the power
supply to stop the run.
16. While your gel is running, obtain and prepare two tubes for 
your transformation as directed in Section F below.
17. After electrophoresis ends, remove the gel cassette from 
the apparatus.
19
Developed by Ann Sliski and Karen Malatesta
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Princeton University
18. Place the cassette on top of the UV transilluminator and  The camera should be set to 4.5 (f
stop) and 2 (an exposure time of
take two photographs of your gel, one for each partner.
1/2 sec). If a lighter exposure is
needed, set the camera to 4.5 and
1 (exposure time of 1 sec).
F. Transformation of DH5 with Your Ligation Reaction: 
The next step in cloning is to incubate your ligation reaction
with the competent DH5 cells you prepared earlier.
1. Obtain two clear 1.7 ml microfuge tubes and three LB + Amp  These tubes are for your
+ X-Gal + IPTG plates (one black, one green, one light blue, and  transformation mixes.
one dark blue stripe on side) from the bench at the front of the  Label two plates “Lig” and the third
lab.
“No DNA”.
2. Use a black marker to label the top of one tube with your 
initials, 7/11 (the date) and “Lig” for ligation mix.
3. Label the top of the second tube with “No DNA”, 7/11 and 
your initials.
4. Obtain fresh ice if necessary.

5. Put these two transformation tubes in ice.

6. Add 40 l sterile TE buffer (clear tube, red dot) to the tube you  Keep this tube of TE.
labeled “Lig”.
7. Add all 10 l of your ligation reaction to this tube and flick it 
gently to mix.
8. Add 50 l sterile TE buffer (clear tube, red dot) to the tube you  Do not add anything else to this tube.
labeled “No DNA”.
9. Gently flick your tube of competent DH5 and add 100 l of  Flicking the tube will bring the cells
off the bottom of the tube. Do not
cells to each transformation tube.
vortex these cells.
10. Flick each transformation tube gently with your fingers to  Keep these tubes in ice at all times.
mix.
11. Let these transformation tubes sit on ice for 30 minutes.

12. After the 30 min on ice ends, put both tubes into the 42o C  This heat shock stimulates the cells to
20
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
take up DNA.
water bath for 90 seconds.
13. Use your P1000 to add 1.0 ml sterile LB growth medium (15  Use sterile technique to avoid
contamination.
ml orange-capped tube) to these two tubes and flick gently to
mix the contents.
14. Leave these tubes on your bench at rt for 45 min.
 This incubation allows for expression
of the plasmid’s antibiotic resistance
genes.
G. Plating Your Transformation Mixes to Selective Plates:
When the 45 min incubation ends, follow the instructions below
to plate your cells to the LB + Amp + X-Gal + IPTG plates.
1. Transfer a 150 l aliquot from your “Lig” tube onto each of 
the two LB + Amp/X-Gal/IPTG plates labeled “Lig”.
2. Sterilize your spreader and spread the 150 l liquid across  Touch the spreader to a clear spot on
the plate before using it to spread the
the surface of each plate until all the liquid has been
cells.
absorbed.
3. Transfer 150 l from your “No DNA” tube onto the plate
labeled “No DNA”.
4. Flame your spreader and use it to spread until all the liquid
is absorbed by this plate.
5. Tape your three plates together, write your initials on the 
tape, and incubate these plates upside down overnight at 37o
C.
6. Leave your two microfuge tubes (labeled “Lig” and “No 
DNA”) on your bench overnight until you are sure your
transformation is successful.
21
Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
III. REFERENCES
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Amann, R.I., Ludwig, W. and K.H. Schleifer. 1995. “Phylogenetic Identification and In Situ
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Baker, G.C., Smith, J.J., and D.A. Cowan. 2003. “Review and Re-Analysis of Domain-Specific
16S Primers.” J. Microbiol. Meth. 55: 541 - 555.
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Cole, J.R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., McGarrell, D. M., Garrity, G. M. and
J. M. Tiedje. 2005. “The Ribosomal Database Project (RDP-II): Sequences and Tools for HighThroughput rRNA Analysis.” Nucleic Acids Res. 33: D294 – D296.
Epicentre Biotechnologies (www.epibio.com).
Handelsman, J. 2004. “Metagenomics: Application of Genomics to Uncultured
Microorganisms.” Microbiol. Mol. Biol. Rev. 68: 669 – 685.
Janssen. P.H., Yates, P.S., Grinton, B.E., Taylor, P.M. and M. Sait. 2002. “Improved
Culturability of Soil Bacteria and Isolation in Pure Culture of Novel Members of the Divisions
Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia.” Appl. Environ.
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Kuske, C.R., Barns, S.M. and J.D. Busch. 1997. “Diverse Uncultivated Bacterial Groups from
Soils of the Arid Southwestern United States That Are Present in Many Geographic Regions.”
Appl. Environ. Microbiol. 63: 3614 - 3621.
Milling, A., Smalla, K., Maidl, F.X., Schloter, M. and J.C. Munch. 2004. “Effects of Transgenic
Potatoes with an Altered Starch Composition on the Diversity of Soil and Rhizosphere Bacteria
and Fungi. Plant & Soil 266: 23 – 39.
Rappe, M. S. and S. J. Giovannoni. 2003. “The Uncultured Microbial Majority.” Annu. Rev.
Microbiol. 57: 369 - 394.
Rondon, M.R., August, P.R., Betterman, A.D., Brady, S.F., Grossman, T.H., Liles, M.R.,
Loiacono, K.A., Lynch, B.A., MacNeil, I.A., Minor, C., Tiong, C.L., Gilman, M., Osburne,
M.S., Clardy, J., Handelsman, J., and R.M. Goodman. 2000. “Cloning the Soil Metagenome: A
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Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University
Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.”
Appl. Environ. Microbiol. 66: 2541 – 2547.
Schloss, P. D. and J. Handelsman. 2004. “Status of the Microbial Census.” Microbiol Mol. Biol.
Rev. 68(4): 686 - 691.
White, T.J., Bruns, T., Lee, S. and J. Taylor. 1990. “Amplification and Direct Sequencing of
Fungal Ribosomal RNA Genes for Phylogenetics.” In PCR Protocols, A Guide to Methods and
Applications, pp. 315 – 322. Edited by M. Innis, D.H. Gelfand, T.J. Sninsky and T.J. White.
Academic Press. San Diego, California.
Woese, C. R. and G. E. Fox. 1977. “Phylogenetic Structure of the Prokaryotic Domain: The
Primary Kingdoms.” Proc. Natl. Acad. Sci. USA. 74: 5088 - 5090.
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Princeton University
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Developed by Ann Sliski and Karen Malatesta
Supported by HHMI, Dept. of Molecular Biology, Council on Science and Technology & Freshman Seminar Program
Princeton University