1. home and garden
2. pest control

Bi 1x Spring 2015: The Protists of the Termite Gut
Special thanks to Jared Leadbetter and Adam Rosenthal
for their time in making this module possible.
1
Overview
In this experiment, we will explore the various protist fauna living in Pacific dampwood termites!
This process will take several weeks, but at the end of the experiment, you will know some of the
protozoa found in termites, and how their presence changes when the termites’ diets change from
cellulose to starch. Your TAs have separated the termite colonies into wood-fed, cellulose-fed and
starch-fed groups. You will begin by extracting DNA from the eukaryotic protists living in the gut
of termites from the three feeding groups. Once this DNA has been extracted, you will use PCR to
amplify a region shared by many types of eukaryotic organisms—the 18S ribosomal RNA gene. After
the PCR reaction, you will clean up the reaction from all enzymes using a commercial kit (Qiagen),
leaving just amplified DNA. A TOPO cloning reaction will be used to separate each 18S gene from the
mixed gene pool of amplified 18S sequences. This will allow us to send our samples in for sequencing,
since a heterogeneous mixture of DNA sequences cannot be sequenced. After sequencing results are
sent back from the sequencing company, we will use the on-line databases to compare the sequences
against those reported by other researchers to determine the protozoa from which we extracted DNA.
Essentially, in this lab, you will perform the process necessary to go from a raw sample (from the
termite gut) to a sequence we can actually analyze and examine!
2
2.1
Background
A Brief Discussion on Termites
Termites are a fascinating example of symbiosis and communal interdependence. In the early twentieth
century, Lemuel Roscoe Cleveland, a professor of zoology, studied the relation between the termites
and the intestinal protozoa that these termites harbored by feeding some groups with wood and others
with cellulose (PNAS, 1923). Cleveland noted that the food would travel unprocessed to the termites’
gut, where it would be broken down by the various protists living there. When the termites were
deprived of wood for more than five days, the wood-ingesting protozoa would die; even when the
termites were returned to a wood diet after this time, the loss of the necessary microbes led to their
death. Interestingly, Cleveland also determined that worker termites that lost the wood-ingesting
protozoa would regain these protozoa when reintroduced to wood alongside workers that had the
necessary protozoa. While both the workers and soldiers housed the protozoa that digested the wood,
only the workers could break down the wood while the soldiers would consume the formers’ fecal
matter. Cleveland’s research implied a symbiotic relationship between termites and the protists in
their guts, as well as the dependence among the termites at large.
2.2
Closely Examining the Protozoa
Like many multicellular organisms, termites rely on various microbes to survive. The microbes in
termite guts break down the wood and produce starch, the main source of energy for these insects. In
this module, we will feed some termites with wood, some with cellulose, and others with pure starch.
It is currently unknown how changing the termites’ diet affects the distribution of protists within the
gut. We will approach this question by extracting DNA from these protists after the termites have
lived on their respective food sources for a sufficient amount of time.
1
Once we have isolated the protozoa genomic DNA, we can determine which protists comprise the gene
pool by resorting to a universal barcode that every protist carries, the ribosomal RNA gene. This
gene is highly conserved across the protist world (and the wider biological world for that matter), and
variations in its sequence can be used to identify the microbes that harbor it. In building phylogenetic
trees, the evolutionary relatedness of different protozoa is judged by how closely their 18S ribosomal
RNA sequences match up (See Figure 1).
Figure 1: Structure of 18S RNA sequence. The sequence is depicted in the shape of its dominant
secondary structure, defined by the list of base pairs and bonds between nucleotides. Recall
that RNA is single-stranded, so it can fold back onto itself and have self-pairings. The variable
regions are used for determining the evolutionary relationships of organisms, whereas the conserved
regions are used for primer design. (Ding et al., Nature 2014)
18S rRNA is a highly conserved region of the ribosomal RNA commonly found in eukaryotic organisms.
2
Due to evolution, different organisms with a common ancestor will show slight variations in their 18S
rRNA gene sequences. By examining the variations in the 18S rRNA DNA sequence, scientists can
determine the evolutionary relationships between various organisms (Field 1988). By analyzing the
18S rRNA from the various protists, we can use existing databases to discern the protists in the termite
gut under each feeding condition.
2.3
Amplifying the 18S RNA
Molecular biologists have designed generic primers to amplify the DNA sequence associated with
18S. We will use a premixed enzyme solution containing a polymerase (machinery involved in DNA
replication from a double stranded template), dNTPs (DNA nucleotides that are input for amplification
of DNA from a template sequence), and MgCl2 . We will also add our template DNA, which is the
protozoa DNA you extracted previously. Your TAs will instruct you on the temperature cycles to
impose on these PCR reactions, and the exact recipe for making the PCR reaction mix to maximize
amplification efficiency. Briefly, the thermocycling process can be described in three steps:
1. Denaturation of double-stranded template DNA into two single stranded template DNA molecules
at high temperature.
2. 18S primer annealing to the single stranded template and the start of polymerase activity at
double-stranded sites at lower temperature.
3. Elongation.
The thermocycler ensures that this three-step process is repeated 30 times such that the template is
exponentially amplified (See Figure 2). After PCR amplification of the 18S DNA sequence, we will
clear the reaction of all enzymes using a commercial kit (Qiagen), leaving just amplified DNA.
2.4
Separating the amplified 18S barcodes
Without using next-generation sequencing techniques, it is impossible to yield reliable sequences from
a mixed batch of fragments. Using a reaction called TOPO cloning, we can isolate each 18S gene from
the heterogeneous gene pool of amplified 18S sequences.
The TOPO cloning reaction requires a commercial kit that includes a circular double-stranded DNA
conventionally called a vector (See Figure 3), salt solution, and water. The purpose is to incorporate
each 18S sequence into one of the TOPO vectors.
From there, we will mix this reaction with chemically competent cells (cells that after a heat shock
will suffer pores in their membrane and consequently uptake foreign DNA, such as the TOPO vector).
After cells have been shocked, we will add growth media in which they can repair the damage and
continue to replicate. After an overnight incubation of cells at 37◦ C, we will plate the cells, and
colonies will form. Note that TOPO vector contains both Kanamycin (Kan) and Ampicillin (Amp)
resistance genes. Hence, we will plate cells on plates containing either one of those antibiotics to
ensure that only those bacteria that have been taken up by the TOPO vector can form colonies. Each
colony will have formed from one cell, and therefore will only carry one 18S gene. We will pick several
colonies, run a PCR with TOPO primers to amplify the 18S signal from each colony, and send the
PCR product for sequencing.
Once we receive the sequencing results, we will use the NCBI databases to compare the sequences
against those reported by other researchers. We will pick the protozoa whose 18S sequence best
3
Figure 2: DNA amplification using PCR. Double-stranded templates dissociate into single-stranded
DNA at around 95◦ C. The temperature is then lowered to 55-65◦ C and primers bind to templates,
forming double stranded DNA. The polymerases bind to double stranded sites and elongate the sequence. These steps are then iterated 30 to 40 cycles and the template DNA is copied exponentially.
Figure from Alberts, et al., Molecular Biology of the Cell, 5th Ed., Garland, 2008.
matches the 18S sequence that we pulled out from the termite gut. Later, you will use these sequences
to build a phylogenetic tree to see the evolutionary relationships between the various microorganisms
in the termite gut. We will provide a more detailed description of databases, sequence alignments,
and other bioinformatics tools in coming weeks.
3
Protocol
This lab is spread out over several weeks. Each section describes what we will do that week. During
the first week, we will extract DNA and set up the 18S PCR reaction. During the second week, we
will run our PCR product on a gel, purify it, and perform the TOPO cloning reaction. In the third
week, we will perform colony PCR (which we will do concurrently for the Luria-Delbr¨
uck experiment).
Finally, a couple of weeks later, we will send out the sequencing results and analyze them.
3.1
Visual Inspection of the Termite Gut
As you learned several weeks ago, the gut of the termite is far from boring sludge. You observed the
enormous Trychonympha chowing down on wood particles, the highly-mobile Trichimotopsis aiding
in the cellulose digestion, and the enigmatic Streblomastix. Before analyzing any sequencing information, you should observe the termite gut under 10x, 40x, and 100x magnification to make qualitative
measurements of how a change in diet affects the distribution of the aforementioned protists. Your
TAs will have prepared three slides of extracted termite gut fluid from each diet. Observe each slide
under the microscope and record your observations. How has the distribution changed? Are there any
protists that dropped in abundance, raised, or stayed the same?
4
=5>?!$+-+/+5/+3-$>363!"#$%&'&()&$*(+,+-.$)+/&
0#$*(+,+-.$)+/&
@AB
!"!"!"##""$"!"#!%"%#"$!!"%#"%%"!$#!!""#!%!"$#""%%""!!!$%!"!%"""##
#%#%#%!!%%$%#%!#"%"!%$##%"!%""%#$!##%%!#"#%$!%%""%%###$"#%#"%%%!!
@-B
"
)"#
#"!%"#%!!%$#!"##%%%""$"!#""%%!#!$!!%%$$$$$$$$$$$"###!$#""%%!#!##
" )87>C3D
!%#"%!"##"$!#%!!"""%%$%#!%%""#!#$##"$$$$$$$$$$$%%!!!#$!%%""#!#!!
*BB
!!#!%"""%%$!""%%!#!!!$%"%"#%#"#%$!#%"%%"!""$%%!"!%##!!$#%!#%%%%"!
##!#"%%%""$#%%""#!###$"%"%!"!%!"$#!"%""%#%%$""#%#"!!##$!"#!""""%#
&'($<
)+%$<
)*($<
!"#%$<
!"#%$<
01$*(+,+-.$)+/&
!"#$23(45(6$789:;$*(+,+-.$)+/&
<93=!&
33>
?
!6"
782
) 493
$#%$<
!"#$%&'()($
425
01
!2
3
24
!"##$%&'()"*(+!,-./01213456(%789$"&:;$'
:9
5
9
1;
32
5
*+,-./!
!"#!"#$%$&'#!#'()$*+!,-.'.!/0/12
!!!!!345!,)*6)*(!.)&'+!,-.'.!78019/
!!!!!:;4!"$<=%'#-.'!,)*6)*(!.)&'+!,-.'.!19901>?
!!!!!@-A!#'"#'..$#!,)*6)*(!.)&'+!,-.'.!1>70177
FigureB&-#&!$C!&#-*.A#)"&)$*+!,-.'!1>7
3: TOPO vector. The insert will be incorporated into the bacterial genome just before the
start ofD19!:'E'#.'!"#)%)*(!.)&'+!,-.'.!/F80//1
LacZ gene. The vector has two antibiotic genes so that bacteria that contain this vector
will be@-AG!0##$%!('*'!CH.)$*+!,-.'.!/1>0?1F
able to grow in the presence of both antibiotics. This figure is from the documentation
!!!!!@-AG!!"$#&)$*!$C!CH.)$*+!,-.'.!/1>0I7>
accompanying
the TOPO kits provided by the vendor, Life Technologies.
!!!!!##$J!"$#&)$*!$C!CH.)$*+!,-.'.!8F?0?1F
K9!"#)%)*(!.)&'+!,-.'.!/I90/2/
KL5L-!3<$*)*(!.)&'+!,-.'.!/7I0/78
3.2 Termite
Gut and DNA Extraction (Week 5)
K>!"#)%)*(!.)&'+!,-.'.!9/?09I>
D19!M$#N-#6!O0/FP!"#)%)*(!.)&'+!,-.'.!98809>F
Today you will
use a commercial kit, MoBio’s PowerSoil, to break open the protozoa that live in
Q-*-%=A)*!"#$%$&'#+!,-.'.!1F/101F>F
termite gut Q-*-%=A)*!#'.).&-*A'!('*'+!,-.'.!118701789
and to isolate and purify the DNA from those cells.
4%")A)<<)*!O&!"P!#'.).&-*A'!('*'+!,-.'.!//F909F29!OAP
4%")A)<<)*!O&!"P!"#$%$&'#+!,-.'.!9F2I0912F!OAP
"R3!$#)()*+!,-.'.!912109?9I
3.2.1 Termite
Gut Extraction
OAP!S!A$%"<'%'*&-#=!.&#-*6
the
1. To begin, you will need to extract the gut fluid from the termites without harvesting any stray
termite cells. You and your partner will receive a petri dish of about five or six termites on ice.
The dish will be labeled with the diet that the termites are on. Make sure to record this in your
notebook.
2. Once the termites are sufficiently subdued, remove the petri dishes from the bucket and place
one of the termites onto parafilm under the dissection microscope. Using a pair of blunt-end
tweezers, gently hold the head of the termite. With a pair of fine-pointed tweezers in the other
hand, gently grab the last few segments of the termite’s rear end. Gently pull with the finepointed tweezers. You should find that the gut should slide out smoothly from the termite’s
exterior.
3. Let the gut dry out for a few seconds to a minute. This will allow the gut to be easily punctured.
4. With a 10 µL pipette ready, take an 18-gauge needle attached to a syringe and gently poke the
gut of the termite. Do not suck up any of the fluid with the syringe; you are only
making a cut with the needle. After the gut has been emptied of fluid, move the gut to
5
the side. Remove the protozoa in the gut with the pipette and place into an Eppendorf tube.
Repeat this with the other termites on the same diet and using the same tube. It is critical
that you do not suck up any termite intestinal material. Place the solution from the
termite gut into a ready solution (Take care not to puncture the parafilm. If you do,
simply use a new piece of film before de-gutting the next termite). Be sure to label
each of your three tubes with the diet.
3.2.2
DNA Extraction
MoBio did an excellent job describing their protocol including descriptions of why each step is performed. However, we will be side-stepping some parts of its protocol, so below is the protocol with
our modifications included. The original MoBio protocol is included at the end of the document if
you are interested. As there are many intricate steps in the extraction process, it is necessary that
we do the step-by-step process as a class. Your TAs will lead you through each step so that we can
assure everyone successfully extracts DNA as we will continue to work with your purified DNA product
over the next few weeks. After the extraction, the concentration of the DNA will be found using a
NanoDrop Spectrophotometer.
1. Add the collection of termite gut fluid into the tube labeled Buffer C0. This buffer is meant
to protect nucleic acids from degradation.
2. Gently vortex to mix for 30 seconds to 1 minute.
3. Add 60 µL of Solution C1 and secure the tube horizontally on a flat-bed vortex pad with tape.
Vortex at maximum speed for 10 minutes. This will complete homogenization and cell lysis.
4. Centrifuge your tube at 10,000 × g (rcf ) for 30 seconds at room temperate. Ensure that the
centrifuge is balanced correctly before beginning the spin.
5. Transfer the supernatant to a clean 2 mL Collection Tube.
6. Add 250 µL of Solution C2 and vortex for 5 seconds. Incubate on ice for 5 minutes. This
solution helps to separate non-DNA substances from the DNA itself.
7. Centrifuge the tubes at room temperate for 1 minute at 10,000 × g.
8. Transfer up to 600 µL of the supernatant to a clean 2 mL Collection tube (avoid the pellets that
are on the side of the tube due to centrifugation).
9. Add 200 µL of Solution C3 and vortex briefly. Incubate on ice for 5 minutes. The solution
similarly precipitates other non-DNA materials.
10. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
11. Transfer up to 750 µL of supernatant to a clean 2 mL collection tube.
12. Shake to mix Solution C4 before you use it. Add 1.2 mL of Solution C4 to the supernatant
((Be careful that the solution doesn’t exceed the rim of the tube) and vortex for 5 seconds.
Solution C4 is a high concentration salt solution. It allows the DNA to bind to the spin filters
that you will use.
13. Load approximately 675 µL onto a Spin Filter and centrifuge at 10,000 × g for 1 minute at
room temperature. Discard any materials that pass through the filter by removing the filter,
then return the filter and add an additional 675 µL of supernatant to the Spin Filter and
centrifuge at 10,000 × g for 1 minute at room temperature. Discard the flow through. Load
6
the remaining supernatant onto the filter and centrifuge at 10,000 × g for 1 minute at room
temperature. Discard the flow through again. During this process the DNA binds to the filter
while contaminants pass through the filter membrane, leaving only DNA.
14. Add 500 µL of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 × g.
Solution C5 is an ethanol based wash solution that removes any additional residual contaminants
while keeping the DNA precipitated onto the filter. Discard the flow through.
15. Centrifuge at room temperature for 1 minute at 10,000 × g. This removes any residual Solution
C5 from the filter.
16. Carefully place the Spin Filter in a clean 2 mL collection tube. Avoiding splashing any Solution
C5 onto the spin filter, as we want to minimize the amount of traces of the wash.
17. Add 40 µL of Solution C6 to the center of the white filter membrane. Solution C6 wets the
entire membrane, making it easier to remove the DNA from the membrane. While the solution
passes through the membrane, DNA in the salt-concentrated environment is released from the
membrane into the salt-free solution.
18. Centrifuge at room temperature for 30 seconds at 10,000 × g.
19. Discard the Spin Filter. The DNA in the tube is now ready for any downstream application.
20. When complete, bring your sample to the TA at the NanoDrop reader, and record the concentration. You will need this for setting up the PCR reaction.
3.3
Amplification of 18S ribosomal DNA by PCR (Week 5)
1. Combine mixes below in PCR tubes. Based on your measured concentration of DNA, you will
have to fill in the blank boxes. If your template DNA is too concentrated to get 1-10 ng, dilute
it as necessary. Ask a TA if you are unsure about this.
Table 1: PCR reaction mixes for 18S amplification
Reagent
No-template
control
No-primer control
Experiment
10.5 µL
sterile water
forward primer
—
1 µL
1 µL
reverse primer
—
1 µL
1 µL
template DNA
(1-10 ng)
—
Perfecta 2×
Mastermix
12.5 µL
12.5 µL
12.5 µL
Total
25 µL
25 µL
25 µL
7
2. Make sure you label your tubes so that you can recognize them! Mix gently, spin down, and
place your tubes in a thermocycler. The PCR reaction conditions are given in the following
table.
Table 2: PCR cycle set up
Step
initial denaturation
denaturation
annealing
extension
final extension
Temperature
95◦ C
95◦ C
55◦ C
72◦ C
72◦ C
Time
3 min.
30 sec.
30 sec.
1 min. 30 s
7 min
Cycles
1
35
1
3. After cycling, the reaction will be maintained at 4◦ C and stored at −20◦ C by the TAs.
3.4
Gel electrophoresis of purified/amplified DNA (Week 6)
1. Form a group of 3 with 2 other people. You will run all of your products on the same gel.
2. Choose one member of your group to prepare the 100bp ladder, and another to prepare the 1kb
ladder. You only need one of each of these per group. Prepare the ladder mixes in the order
given by the table below.
Table 3: Ladder mixes for gel electrophoresis
Reagent
sterile water
ladder
loading dye (6×)
total
100 bp ladder mix
4 µL
1 µL
1 µL
6 µL
1 kbp ladder mix
4 µL
1 µL
1 µL
6 µL
3. Each group will prepare a sample from each PCR reaction from last week to be analyzed by
electrophoresis. This will check whether there was any contamination, as the no-primer and
no-template controls should not have amplified anything. Prepare the following PCR product
mixes for loading onto the gel.
Table 4: PCR reaction mixes for gel electrophoresis
Reagent
No-primer control
DNA
Loading dye (6×)
Total
5 µL
1 µL
6 µL
No-template
control
5 µL
1 µL
6 µL
Experiment
5 µL
1 µL
6 µL
4. Load your samples onto a gel. There are 15 lanes total, and they should be laid out as follows.
8
Table 5: Lane assignments for gel electrophoresis
Lane
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Contents
100 bp ladder mix
empty
group 1: no-primer control
group 1: no-template control
group 1: experiment
empty
group 2: no-primer control
group 2: no-template control
group 2: experiment
empty
group 3: no-primer control
group 3: no-template control
group 3: experiment
1 kbp ladder mix
empty
5. The gel is a 1% agarose gel, with SYBR Safe for viewing the DNA. It will be run at 100V for 30
min. The expected band size is ∼ 1.8 kb. The ladders you are using are pictured below. While
your gel is running, begin the purification of your experimental PCR reaction.
Figure 4: Ladders for use in gel electrophoresis. The left ladder is the 100 bp ladder and the right
is the 1 kbp ladder. The images come from the product information websites of the vendors, New
England Biosystems and Life Technologies, respectively.
3.5
PCR purification (Week 6)
We will use kits made by Qiagen to purify our PCR products.
1. Add 5 × the amount of PCR sample in the form of Buffer PB to your PCR sample and mix by
pipetting. For example, if you had 10 µL of PCR sample, you would add 50 µL of Buffer PB.
Add 5µL of 3M Na-Acetate pH 5.0. Upon addition, mix the tube by inverting.
2. Place a QIAquick spin column in a purple 2 mL collection tube.
3. To bind DNA, apply the sample to the QIAquick spin column and centrifuge at 13,000 rpm for
1 min.
9
4. Discard flow-through into the provided liquid waste container and place the QIAquick column
back in the same tube.
5. To wash add 0.75 mL Buffer PE to the QIAquick column and centrifuge at 13,000 rpm for 1
min.
6. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the
column at 13,000 rpm for an additional 1 min.
7. Place the QIAquick column in a clean 1.5 mL microcentrifuge tube.
8. To elute DNA, place 30 µL of Buffer EB to the center of the QIAquick membrane (but be careful
not to puncture it) and let column stand for 5 minutes. Centrifuge the column at 13,000 rpm
for 1 min.
9. Be sure that your tube is clearly labeled!
3.6
TOPO reactions (Week 6)
We will use kits from Life Technologies for the TOPO cloning step. The kits come with chemically
competent cells, which means their membranes will become porous upon heat treatment, as opposed
to electrical shock, which is commonly used.
1. Prepare a solution for the TOPO reaction to ligate your PCR product into the TOPO vector
according to the table below.
Table 6: TOPO reaction mixture
Reagent
sterile water
purified PCR product
salt solution
TOPO vector
Total
Volume
3 µL
1 µL
1 µL
1 µL
6 µL
2. Mix the reaction gently and incubate the TOPO reaction for 10 minutes at room temperature.
3. Place the reaction on ice.
4. Add 2 µL of the TOPO Cloning reaction to a vial of TOP10 cells that have been thawed on ice.
5. Mix gently by swirling with the pipette tip. Do not mix by pipetting up and down!
6. Incubate on ice for 30 minutes.
7. Heat-shock the cells for 2 minutes at 42◦ C without shaking.
8. Add 250 µL of room temperature S.O.C. medium.
9. Cap the tube tightly and shake the tube horizontally (200rpm) at 37◦ . This step will allow the
bacterium to express the antibiotic resistance. After one hour of recovery at 37C, plate 100µL
onto LB Kanamycin plates and place in the incubator.
10. The TAs will store your plates at 4◦ for later use.
10
3.7
Colony PCR (Week 7)
In this protocol, you will be performing steps for both the Luria-Delbr¨
uck and Termite Gut experiments. The process for setting them up to prepare for sequencing is very similar.
1. Before combining your colony PCR reaction mixes, you will need to suspend colonies in 50 µL of
sterile water (provided). Suspend 1 colony of wild type cells from the Luria-Delbr¨
uck experiment
in 50 µL of water. Repeat this process for 1 colony of mutator cells from the Luria-Delbr¨
uck
experiment.
2. Pick 3 colonies from your TOPO reaction. Suspend each one in its own 50 µL of water. The
two types of colonies (L-D vs. TOPO) will be amplified using different primers, so keep this
in mind when you perform your mixes. We will use CAN1 primers for yeast and M13 primers
for the TOPO sequences. You will have a total of 5 “experiment” PCR reactions (three TOPO
colonies, a wild-type yeast colony, and a mutator yeast colony) and 2 no-template controls (one
for the TOPO reactions and one for Luria-Delbr¨
uck). The recipes for the PCR mixes are shown
below.
Table 7: Colony PCR reaction mixes
Reagent
sterile water
Phusion 2× Mastermix
forward primer (5 µM)
reverse primer (5 µM)
colony suspension
total
No-template control
10.5 µL
12.5 µL
1 µL
1 µL
—
25 µL
Experiment
10 µL
12.5 µL
1 µL
1 µL
0.5 µL
25 µL
3. Mix gently, spin down, and place tubes in a thermocycler. Clearly label your tubes! (This may
be easier on the side of the tube) The PCR reaction conditions are given in the following table.
Table 8: Thermocycler conditions for colony PCR using M13 and CAN1 primers
Step
initial denaturation
denaturation
annealing
extension
final extension
Temperature
98◦ C
98◦ C
51◦ C
72◦ C
72◦ C
Time
30 sec.
8 sec.
20 sec.
45 sec.
5 min
Cycles
1
30
1
4. After cycling, the reaction will automatically be maintained at 4◦ C until you retrieve it for gel
electrophoresis.
3.8
Gel electrophoresis of colony PCR products (Week 7)
• Form a group of 2 with your lab partner. You will run all of your products on the same gel.
• Each person will prepare 7 samples for loading—one from each PCR reaction. The ladder has
been premixed for you. This will check whether there was any contamination, as the no primer
and no template controls should not have amplified anything. Each sample will consist of 1 µL
of 6× loading dye and 5 µL of PCR products, as per table 4.
11
• There are 15 lanes total, and the lanes should be laid out as follows.
Table 9: Lane assignments for gel electrophoresis of
colony PCR products
Lane
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Contents
100 kbp ladder mix
group 1: Luria-Delbr¨
uck
no-template control
group 1: Luria-Delbr¨
uck wild type
group 1: Luria-Delbr¨
uck mutator
group 1: TOPO no-template
control
group 1: TOPO reaction 1
group 1: TOPO reaction 2
group 1: TOPO reaction 3
group 2: Luria-Delbr¨
uck
no-template control
group 2: Luria-Delbr¨
uck wild type
group 2: Luria-Delbr¨
uck mutator
group 2: TOPO no-template
control
group 2: TOPO reaction 1
group 2: TOPO reaction 2
group 2: TOPO reaction 3
• The gel is a 1% agarose gel, with SYBR Safe for viewing the DNA. It will be run at 100V for
30 min. The expected band size is around 1300 bp.
• The unpurified products will be sent to Laragen (a Culver City-based sequencing company) for
sequencing. The company will perform the purification step we did previously, as well as the
sequencing.
3.9
Bioinformatics (Week 9)
We will have a separate tutorial for bioinformatics during week 9.
4
Assignment
Problem 0 (Summary).
Write a summary of this experiment, its objectives, and conclusions between one paragraph and one
page in length.
Problem 1 (Review of molecular biology techniques).
In this experiment, we have extracted DNA, amplified it using PCR, and analyzed it using gel electrophoresis. In this problem, we will review these concepts.
a) Explain how PCR works. Go through each step and describe what is happening to the DNA
strands.
12
b) Why do we want only a single band on our gels after PCR (besides the lanes with ladder)?
c) Why was the TOPO cloning step necessary?
d) Why were the transformed cells plated onto plates with Kanamycin and LB, as opposed to just
LB?
Problem 2 (Visual inspection of gut contents).
In lab, you looked at the gut contents of termites that were fed their native diet of ponderosa pine,
a diet of pure cellulose, and a diet of pure starch. Additionally, we provided you with representative
images of these gut contents. Comment on differences you observed among the different diets. Are
there species of protist that are absent under some dietary conditions? Does the density of protists in
the gut vary with diet? Comment on any other observations you may have. Hypothesize as to why
you see the differences you do.
Problem 3 (Phylogeny of the termite gut).
You were provided with file trychonympha_and_tricomitopsis.fna and streblomastix_18S.fna.
Align the sequences in these file and build a phylogenetic tree. Be sure to mention what phylogeny
algorithm you used. Note: Because Streblomastix is so different from the other species, it might help
to make a tree omitting Streblomastix to make it easier to see the other species.
Comment on the genetic difference between the major branches on your phylogenetic tree. If we
make the gross oversimplification that the mutation rate is 0.1% per million years, how long have the
respective branches been diverging? Be sure to include a printout of your tree(s). Hint: The scale
bar in the phyologenetic tree represents the amount of change per nucleotide. This means that the
horizonal scale confers meaning. The vertical scale does not.
Problem 4 (Analysis of the 18S sequences).
a) Provide a brief discussion as to why we perform analysis of the gene sequences when studying
the effect of diet on the microbiome. (You may be interested in this article, if you are interested
in the effects of diet on your own gut organisms.)
b) Why do we use 18S as the gene that we sequence?
c) Prior to doing alignment for our experimental sequences, we need to trim the sequences and
also do paired-end joining. Because not all of you have scikit-bio, we have provided three
FASTA files with the trimmed, joined sequences, wood_18s.fasta, cellulose_18s.fasta, and
starch_18s.fasta. These files were generated in the bioinformatics tutorial. Go through the
last code block of that tutorial and describe what the code does. This is to ensure that you
understand what you need to do to perpare the samples for use in database searches.
d) Using the files described in part (c), perform BLAST searchs to identify where the sequences
you extracted come from. Discuss what species the samples belong to and compare this to what
you saw visually in the microscope.
e) Build a phylogenetic tree using the experimental sequences. You should not include sequences
that your BLAST search revealed to be the result of contanimation. Comment on this phylogenetic tree in light of the species you identified from your BLAST search and with respect to the
13
tree you generated in problem 3. Does this help shed light on how the termite diet affects what
organisms live in its guts?
Problem 5 (Data and code).
Attach all code, data, and sample images not specifically asked for in the other problems.
Appendix: Extraction protocol from MolBio
The DNA extraction protocol from MoBio is on the following pages. Note that this is not the
protocol we will use in lab, since we have some modifications to it, but is here for reference.
14
Detailed Protocol (Describes what is happening at each step)
Please wear gloves at all times
1. To the PowerBead Tubes provided, add 0.25 grams of soil sample.
What’s happening: After your sample has been loaded into the PowerBead Tube, the next step is a
homogenization and lysis procedure. The PowerBead Tube contains a buffer that will (a) help disperse the soil
particles, (b) begin to dissolve humic acids and (c) protect nucleic acids from degradation.
2. Gently vortex to mix.
What’s happening: Gentle vortexing mixes the components in the PowerBead Tube and begins to disperse the
sample in the PowerBead Solution.
3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60 C until the precipitate has
dissolved before use.
What’s happening: Solution C1 contains SDS and other disruption agents required for complete cell lysis. In
addition to aiding in cell lysis, SDS is an anionic detergent that breaks down fatty acids and lipids associated
with the cell membrane of several organisms. If it gets cold, it will form a white precipitate in the bottle. Heating
to 60 C will dissolve the SDS and will not harm the SDS or the other disruption agents. Solution C1 can be
used while it is still warm.
4. Add 60 l of Solution C1 and invert several times or vortex briefly.
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex
(MO BIO Catalog# 13000-V1-24) or secure tubes horizontally on a flat-bed vortex pad with tape.
Vortex at maximum speed for 10 minutes.
Note: If you are using the 24 place Vortex Adapter for more than 12 preps, increase the
vortex time by 5-10 minutes.
Note: 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 introduced at this step.
By randomly shaking the beads in the presence of disruption agents, collision of the beads with
microbial cells will cause the cells to break open.
What’s happening: The MO BIO Vortex Adapter is designed to be a simple platform to facilitate keeping the
tubes tightly attached to the vortex. It should be noted that although you can attach tubes with tape, often the
tape becomes loose and not all tubes will shake evenly or efficiently. This may lead to inconsistent results or
lower yields. Therefore, the use of the MO BIO Vortex Adapter is a highly recommended and cost effective way
to obtain maximum DNA yields.
6. Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing. Centrifuge tubes at
10,000 x g for 30 seconds at room temperature. CAUTION: Be sure not to exceed 10,000 x g or tubes
may break.
7. Transfer the supernatant to a clean 2 ml Collection Tube (provided).
Note: Expect between 400 to 500 l 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 soil particles. The
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
8
presence of carry over soil or a dark color in the mixture is expected in many soil types at this step.
Subsequent steps in the protocol will remove both carry over soil and coloration of the mixture.
8. Add 250 l of Solution C2 and vortex for 5 seconds. Incubate at 4 C for 5 minutes.
®
What’s happening: Solution C2 is patented Inhibitor Removal Technology (IRT). It contains a reagent to
precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins. It is
important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit
downstream DNA applications.
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
10. Avoiding the pellet, transfer up to 600 l of supernatant to a clean 2 ml Collection Tube (provided).
What’s happening: The pellet at this point contains non-DNA organic and inorganic material including humic
acid, cell debris, and proteins. For the best DNA yields, and quality, avoid transferring any of the pellet.
11. Add 200 l of Solution C3 and vortex briefly. Incubate at 4 C for 5 minutes.
®
What’s happening: Solution C3 is patented Inhibitor Removal Technology (IRT) and is a second reagent to
precipitate additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins. It
is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit
downstream DNA applications.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
13. Transfer up to 750 l of supernatant to a clean 2 ml Collection Tube (provided).
What’s happening: The pellet at this point contains additional non-DNA organic and inorganic material
including humic acid, cell debris, and proteins. For the best DNA yields, and quality, avoid transferring any of
the pellet.
14. Shake to mix Solution C4 before use. Add 1.2 ml of Solution C4 to the supernatant (be careful
solution doesn’t exceed rim of 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 concentrations to allow binding of DNA, but not nonDNA organic and inorganic material that may still be present at low levels, to the Spin Filters.
15. Load approximately 675 l onto a Spin Filter and centrifuge at 10,000 x g for 1 minute at room
temperature. Discard the flow through and add an additional 675 l of supernatant to the Spin Filter
and centrifuge at 10,000 x g for 1 minute at room temperature. Load the remaining supernatant onto
the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature.
Note: A total of three loads for each sample processed are required.
What’s happening: 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 membrane.
16. Add 500 l of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g.
What’s happening: Solution C5 is an ethanol based wash solution used to further clean the DNA that is bound
to the silica filter membrane in the Spin Filter. This wash solution removes residual salt, humic acid, and other
contaminants while allowing the DNA to stay bound to the silica membrane.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
9
17. Discard the flow through from the 2 ml Collection Tube.
What’s happening: This flow through fraction is just non-DNA organic and inorganic waste removed from the
silica Spin Filter membrane by the ethanol wash solution.
18. Centrifuge at room temperature for 1 minute at 10,000 x g.
What’s happening: This second spin removes residual Solution C5 (ethanol wash solution). It is critical to
remove all traces of wash solution because the ethanol in Solution C5 can interfere with many downstream
DNA applications such as PCR, restriction digests, and gel electrophoresis.
19. Carefully place Spin Filter in a clean 2 ml Collection Tube (provided). Avoid splashing any Solution
C5 onto the Spin Filter.
Note: It is important to avoid any traces of the ethanol based wash solution.
20. Add 100 l of Solution C6 to the center of the white filter membrane.
Note: Placing the Solution C6 (sterile elution buffer) in the center of the small white membrane will
make sure the entire membrane is wetted. This will result in a more efficient and complete release
of the DNA from the silica Spin 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) which lacks salt.
Alternatively, sterile DNA-Free PCR Grade Water may be used for DNA elution from the silica Spin
Filter membrane at this step (MO BIO Catalog# 17000-10). Solution C6 contains no EDTA. If
DNA degradation is a concern, Sterile TE may also be used instead of Solution C6 for elution of
DNA from the Spin Filter.
21. Centrifuge at room temperature for 30 seconds at 10,000 x g.
22. Discard the Spin Filter. The DNA in the tube is now ready for any downstream application. No
further steps are required.
We recommend storing DNA frozen (-20 to -80 C). Solution C6 does not contain any EDTA. To
concentrate DNA see the Hints & Troubleshooting Guide.
Thank you for choosing the PowerSoil® DNA Isolation Kit.
Technical Information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: [email protected] Website: www.mobio.com
10