CULTIVATION INDEPENDENT ANALYSIS OF SOIL BACTERIA PART 1
To add to the growing field of environmental microbiology, students at Delaware Tech are
characterizing soil microbes throughout Delaware and neighboring states. In Principles of
Microbiology, students performed cultivation dependent analysis to isolate bacteria using
standard microbiology techniques. These cultured bacteria were identified by sequencing 16S
ribosomal DNA (rDNA), the genes that encode 16S ribosomal RNA (rRNA). This involved
extracting DNA from the cells, using PCR to amplify the 16S rDNA gene, and sequencing the
amplicon. BLAST is then used to perform a database search to determine the most likely
identity of the individual isolated bacteria.
Typically, only about 1% of the bacterial species present in soil will grow on media in the
laboratory. As part of the on-going research, we wish to know what other bacteria are present.
The easiest way to accomplish this task is to extract total DNA from the soil, amplify all 16S
rRNA genes, and send them for sequencing. However, with most sequencing instruments, an
unreadable chromatogram would be the result.
To have useable sequencing results, the sequences from different species of bacteria need to
be isolated from each other. The method that will be used in this laboratory will be to use
Invitrogen’s TOPO TA cloning kit to clone the 16S rRNA amplicons into plasmid. Competent E.
coli cells are then transformed with the plasmid and colonies grown on plates. Each colony will
contain plasmid with a sequence from one species of bacteria. Mini plasmid preparations will
be performed and the isolated plasmid sent for sequencing. The resulting sequences will then
be analyzed to identify the bacteria present in the soil.
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Flow Chart of Cloning Protocol:
Extract total DNA from soil (Part 1)
↓
PCR to amplify 16S rRNA genes (Part 1)
↓
Agarose gel of amplicons (Part 2)
↓
Purify amplicon from gel (Part 2)
↓
Ligate amplicon into plasmid (Part 2)
↓
Transform competent E. coli with constructs (Part 2)
↓
Pick colonies LB/kanamycin plates (Part 3)
↓
Grow overnight cultures of transformants (Part 3)
↓
Mini plasmid preps (Part 3)
↓
Restriction enzyme digests to confirm presence of insert (Part 3)
↓
Agarose gel (Part 4)
↓
Prepare plasmid for sequencing (Part 4)
↓
Analyze sequences (Part 5)
MATERIALS:
Mo Bio PowerSoil DNA Isolation Kit
Alcohol wipes
Metal spatula
Permanent marker
Milligram balance
Micropipettes
Sterile yellow and blue tips
Vortex
Tape
Microcentrifuge
Ice bath in bucket
Foam float
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PCR tubes
16S rRNA PCR primer 1492R, 10 µM
16S rRNA PCR primer 27F, 10 µM
2x TopTaq PCR buffer
Sterile water
Thermal cycler
5’  3’ Sequence
GGT TAC CTT GTT ACG ACT T
AGA GTT TGA TCM TGG CTC AG
Primer Name
16S_1492R
16S_27F
Position
1492-1510
8-27
Tm (°C) (50 mM KCl)
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56
PROCEDURE:
A. DNA Extraction from soil sample
1. Bleach your benchtop and cover with a piece of absorbent paper.
2. Follow the procedure for the Mo Bio PowerSoil DNA Isolation Kit with the following
modifications.
a. Label your tube with your initials.
b. Step 5. Increase the vortex time to 15 minutes.
c. Step 18. Increase centrifuge time to 2 minutes to ensure complete ethanol
removal.
3. To add soil to the PowerSoil Bead Tube, use a metal spatula that has been cleaned
with an alcohol wipe.
4. Use sterile micropipette tips and microfuge tubes during all procedures.
5. Avoid contaminating the PowerSoil Kit solutions. Depress the micropipette plunger
before placing the micropipette over the solution.
B. PCR Amplification of 16S rDNA
1. Wear gloves when performing all PCR procedures.
2. Label four PCR tubes with 1-4 and your initials.
3. Add the components listed in the following table to each tube. Add the solutions to
the bottom of the tubes. After adding the 2x PCR Mix, pipette gently up and down to
mix.
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Template from soil extraction
PCR primer 1492R (10 μM)
PCR primer 27F (10 μM)
Sterile DI water
2x PCR Mix
Tube 1
9 μL
3 μL
3 μL
0 μL
15 μL
Tube 2
5 μL
3 μL
3 μL
4 μL
15 μL
Tube 3
2 μL
3 μL
3 μL
6 μL
15 μL
Tube 4
0 μL
3 μL
3 μL
9 μL
15 μL
4. Cap the tubes tightly.
5. Place the tubes into a thermal cycler and start the Soil Microbe Program.
a. Hot-start denaturation: 95°C for 5:00 minutes.
b. Three-step cycling repeated 35 times:
Denaturation: 94°C for 1:00 minute
Annealing: 48°C for 30 seconds
Extension: 72°C for 1 minute
c. Final Extension: 72°C for 15 minutes
d. Hold: 4°C for infinite time (∞)
6. Remove the PCR tubes from the thermal cycler and store them at -20°C until the
next lab period.
DISCUSSION QUESTIONS:
1. Besides bacteria, what other microbes are present in the soil sample?
2. How do we avoid amplifying DNA sequences from non-bacteria organisms?
3. There are probably more than 10,000 bacterial species in the soil samples. List two
factors that could influence the chances that the class will be able to clone a particular
species.
4. Bacteria differ in the number of rRNA operons (rrn) in their genomes. To investigate this
difference, go to the RNA list at the J. Craig Venter Institute Comprehensive Microbial
Resource.
http://cmr.jcvi.org/cgi-bin/CMR/shared/MakeFrontPages.cgi?page=rna_list
Select five bacterial genomes to search. Select rRNA for RNA type.
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Bacteria produce three different rRNAs, 5S, 16S, and 23S. The 5S rRNA is approximately 120 bp
in length; the 16S rRNA is approximately 1500 bp in length; and the 23S rRNA is approximately
3000 bp in length. For Acidobacteria bacterium Ellin345 three rRNA sequences are listed. The
numbers in the 5’ and 3’ end columns correspond to the nucleotide positions on the
chromosome where the operons are located. By taking the difference between the two
numbers, we can determine that NT02AB4890 is the 5S gene, 4891 is the 23S gene, and the
4895 is the 16S gene.
a. List your five species of bacteria and how many of each type of rRNA operons.
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b. How do you think the number of rRNA operons correlates to growth rate and
response to growth substrates in the environment? Consider the function of
ribosomes.
c. How will the number of 16S rRNA operons a bacterium possesses affect its chances
that it will be cloned in our procedure?
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CULTIVATION INDEPENDENT ANALYSIS OF SOIL BACTERIA PART 2
The DNA that was extracted from the soil in the first part of the lab contains as many as 10,000
genomes. The presence of these extra sequences may lead to nonspecific priming during the
PCR reaction and the production of undesired amplicons. To reduce the chance that the
nonspecific amplicons will be cloned, the PCR products of the correct size are separated from
other products using agarose gel electrophoresis.
Agarose is a polysaccharide that is extracted from red algae. It is melted in the presence of
electrophoresis buffer and poured into a bed to form a semi-solid matrix. When the gel is
placed in a gel chamber, an electrical current is run through it. Since DNA is negatively charged,
it will migrate towards the anode such that smaller sized DNA molecules will move faster than
larger. The band containing DNA amplicons of the expected size (1510 – 8 = 1502 bp) will be cut
from the gel and extracted using a commercial kit.
Typically, cloning is done with T4 ligase which requires lengthy incubation at low temperatures
for efficient cloning. The Invitrogen TOPO TA kit uses Topoisomerase I from Vaccinia virus
instead of ligase. Type I topoisomerases are enzymes found in all organisms and some viruses
that release negative supercoils from DNA. The kit comes with linear plasmid that has an
overhanging 3’ deoxythymidine (T) with topoisomerase I covalently bound. The topoisomerase
binds to DNA after a specific sequence (5’-CCCTT) in one strand. The energy in the broken
phosphodiester backbone is conserved by formation of a covalent bond between the 3’
phosphate of the cleaved bond and a tyrosine residue of the topoisomerase (see the figure
below). The phospho-tyrosyl bond between the DNA and enzyme can be attacked by the 5’
hydroxyl of the original cleaved strand or the added PCR amplicon, reversing the reaction and
releasing topoisomerase.
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Taq polymerase has a nontemplate-dependent terminal transferase activity, which adds a
single deoxyadenosine (A) to the 3’ ends of PCR products. This makes a small sticky end that will
hybridize with the overhanging 3’ deoxythymidine (T) residues on the linearized vector. This
allows PCR inserts to ligate efficiently with the vector.
The vector used for cloning is pCR®4-TOPO; the map for which is shown below. This plasmid has
the ccdB gene fused to the C-terminus of the LacZα fragment. The ccdB gene is a potent
inhibitor of DNA gyrase, a topoisomerase that is critical for cell growth. Ligation of a PCR
product disrupts expression of the lacZα-ccdB gene fusion, permitting growth of only positive
recombinants upon transformation. Cells that contain non-recombinant vector are killed upon
plating. Therefore, blue/white screening is not required. Periodically, colonies are produced
that contain vector only. These usually result from frameshift events (usually T-T mismatches)
which disrupt the lacZα-ccdB reading frame.
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MATERIALS:
Agarose
50x Tris Acetate EDTA (TAE) Buffer
SYBR Safe
250-mL Erlenmeyer flask
25- or 50-mL Erlenmeyer flask
Permanent marker
Gel electrophoresis chamber and set up
Blue and yellow tips
Micropipettes
25-mL graduated cylinder
1000-mL graduated cylinder
UV light box
Scalpel or razor blade
Qiagen Gel Extraction Kit
Milligram balance
50°C heat block
Isopropanol
Invitrogen TOPO TA Cloning Kit for Sequencing
PCR tube
Competent E. coli cells
Ice bath in ice bucket
S.O.C. medium
42°C heat block
37°C shaking incubator
LB plates containing 75 μg/mL kanamycin
Sterile 3 mm glass beads
PROCEDURE:
A. Agarose Gel Electrophoresis
1. Two groups will share one gel.
2. PCR amplicons will be separated on a 0.8% gel. The large BioRad gels require 90 mL of
melted agarose solution to fill the bed to the right height.
3. Using a weigh boat or weigh paper, measure out 0.72 g agarose and pour into a 250 mL
Erlenmeyer flask.
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4. Using a 100-1000 µL micropipette, add 1.8 mL (two aliquots of 900 µL) 50x TAE buffer to
the Erlenmeyer flask with the agarose.
5. Add 90 mL of deionized water.
6. Swirl the flask gently to disperse the agarose without getting too much of the agarose
on the side of the flask.
7. Mark the side of the flask with a permanent marker at the level of the liquid.
8. Invert a 25- or 50-mL Erlenmeyer flask over the top of the flask containing the agarose.
9. Use a microwave to melt the agarose.
a. Heat initially for 30 to 60 seconds.
b. Using a hot pad, swirl the solution.
c. Continue heating in the microwave for 15 to 30 second increments with swirling
until the agarose is completely melted.
d. Avoid boiling vigorously or the solution may boil over.
e. If the liquid level is below the level of the mark made on the side of the flask, fill
to the line with DI water and swirl the flask.
10. After the agarose has cooled slightly, add 6 µL SYBR Safe, and gently swirl to the flask to
avoid adding air bubbles to the agarose.
11. While the agarose is cooling, prepare the gel bed.
a. Place the tray in the gel caster.
b. Place the edge of the tray against the non-moveable wall.
c. Slide the moveable wall against the edge of the tray.
d. Engage the cam by turning and pressing downward.
e. Place the leveling bubble in the center of the tray.
f. Adjust the leveling feet to center the leveling bubble in the middle of the black
circle.
12. When the agarose has cooled to about 60°C (cool enough to hold with a bare hand),
slowly pour the agarose into the gel bed.
a. Do not pour the agarose too hot; this will cause warping of the gel tray and
caster.
b. If air bubbles are in the agarose, tap the gel bed on the bench top to bring the air
bubbles to the top of the agarose.
c. Place the comb in the first set of notches at one end of the bed.
d. Immediately rinse out the Erlenmeyer flask with warm tap water to avoid
agarose from solidifying in the flask.
e. Wash the flask and place on a drying rack.
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13. Allow the gel to completely cool and solidify which takes about 30 minutes.
14. Add 5 μL 5x loading buffer to each PCR sample.
15. Prepare 750 mL buffer for the gel chamber.
a. Use a 25-mL graduated cylinder to measure 15 mL of 50x TAE buffer.
b. Add the buffer concentrate to a 1000-mL graduated cylinder.
c. Bring to 750 mL with DI water.
d. Pour the buffer into the gel chamber.
16. Once the gel has completely cooled, remove the comb from the gel by pulling straight
up. Avoid tearing the gel.
17. Loosen the cam lever and slide the moveable wall back. Carefully slide the gel tray out
of the caster.
18. Place the gel bed into the buffer chamber. The gel should be covered by buffer.
19. Load the gel.
a. Place 8 μL of 100-bp ladder in the first lane.
b. Load 20 μL of each PCR sample into separate wells; skip wells between samples.
20. Place the cover on the gel and plug the wires into the power supply.
21. Turn on the power supply, and run the gel at 100V for 60 minutes.
22. Turn off the power supply and unplug the wires from the power supply. Remove the
cover from the buffer chamber.
23. Carefully remove the gel bed with gel from the chamber, and take a photograph.
24. Weigh a 2-mL microcentrifuge tube, and record the mass.
25. Place the gel on the UV light box in the instrument room.
26. Using a razor blade or fine scalpel, cut the 1500 bp band from the lane with the
strongest band. Avoid scratching the surface of the light box.
27. Place the piece of agarose into the 2-mL microcentrifuge tube. Weigh the tube plus
agarose, and record the mass.
28. Determine the mass of the gel piece.
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29. Rinse the buffer chamber, gel bed and comb with tap water and then with distilled
water. Do not dry the chamber with a paper towel. Place the cover on the buffer
chamber, and return to the cabinet to dry. Return the gel bed, comb, and caster to the
cabinet.
B. Gel Extraction
1. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional
table-top microcentrifuge.
2. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μL).
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the
tube every 2–3 min to help dissolve the gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow
(similar to Buffer QG without dissolved agarose).
a. If the color of the mixture is orange or violet, add 10 μL 3 M sodium acetate, pH
5.0, and mix.
b. The color of the mixture will turn yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2-ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column (up to a maximum of 800 μL)
and centrifuge for 1 min.
8. Discard flow-through and place the QIAquick column back into the same tube. For
sample volumes of >800 μL, load and spin again.
9. To wash, add 0.75 ml Buffer PE to QIAquick column and let stand for 5 minutes.
10. Centrifuge for 1 min.
11. Discard flow-through and place the QIAquick column back into the same tube.
12. Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 2
min at 17,900 x g (13,000 rpm) to remove residual wash buffer.
13. Place QIAquick column into a clean 1.5-ml microcentrifuge tube.
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14. To elute DNA, add 30 μL Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick
membrane, let stand 2 min and centrifuge the column for 1 min.
15. The amplicon (PCR product) is in the flow through. Label the tube with your initials and
place in an ice bath.
C. TOPO Cloning Reaction
1. In a PCR tube, set up the cloning reaction by adding the following reagents.
4 μL PCR product extracted from gel
1 μL Salt Solution
1 μL TOPO vector
2. One group in the class should set up a negative control ligation mixture containing the
following reagents.
4 μL DI water
1 μL Salt Solution
1 μL TOPO vector
3. Mix reaction gently, and incubate for 15 minutes at room temperature.
4. Prepare an ice bath in an ice bucket.
5. Add all of the TOPO cloning reaction mixture to a vial of competent E. coli, and mix
gently by flicking the tube. Do not mix by pipetting up and down.
6. The group that set up the negative control ligation should add all of the negative control
reaction mixture to a second vial of competent E. coli. Carry out all of the
transformation steps with the negative control.
7. A second group in the class should perform a positive control for transformation with
pUC19 plasmid. Add 10 pg of pUC19 to a vial of competent E. coli. Carry out all of the
transformation steps with the positive control.
8. Incubate on ice for 15 minutes.
9. Heat-shock the cells for 30 seconds at 42°C without shaking.
10. Immediately transfer the tubes back on ice for 1 minute.
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11. Remove the cells from the ice bath and add 250 μL of room temperature S.O.C.
medium.
12. Cap the tube tightly and shake horizontally (200 rpm) at 37°C for 1 hour.
13. Each group should pre-warm five LB + amp plates in the 37°C nonshaking incubator.
14. On one plate, use the sterile beads to spread 50 μL of the cells transformed with vector
+ PCR amplicon.
a. Pour 10 to 12 sterile 3 mm glass beads onto an agar plate. Tip the plate slightly
so that the beads move to the side of the plate.
b. Add 50 μL of the cells transformed with vector plus insert to the center of the
plate.
c. Close the lid of the plate.
d. Keeping the plate horizontal on the bench, gently shake the beads across the
surface of the agar plate. Periodically rotate the plate while shaking.
e. Shake the beads back and forth until the solution has been spread evenly and
the surface of the plate looks dry.
f. Discard the beads in a beaker filled with bleach.
g. Repeat for the second and third plates.
15. On the second plate, spread 100 μL of the transformed cells.
16. On the third plate, spread 150 μL of the transformed cells.
17. On the fourth plate, spread 100 μL of the cells transformed with vector alone (negative
control).
18. On the fifth plate, spread 10 μL of the pUC19 transformed cells plus 20 μL S.O.C.
medium (positive control).
19. Incubate at 37°C for 36 to 48 hours to allow for sufficient growth.
20. Record the approximate number of colonies on each plate.
Your lab report should include discussion about the size of the amplicon, amount of amplicon
relative to background smearing, amount of amplicon in each lane of the gel, quality of the gel,
appearance of the negative control PCR reaction, number of colonies on each plate, and results
of positive and negative controls for transformations.
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CULTIVATION INDEPENDENT ANALYSIS OF SOIL BACTERIA PART 3
Before the recombinant plasmids are submitted for sequencing, a second screening is
performed to confirm that the colonies contain 16s rDNA amplicon cloned into pCR4-TOPO.
Colonies from part 2 are selected, and cultures are grown in broth containing a selective
antibiotic. The plasmids are extracted from the cells using a commercial kit, and then digested
with EcoRI, a restriction endonuclease. Since there are restriction sites for EcoRI on either side
of the cloning site, the DNA insert will be released from the vector. The resulting fragments will
be analyzed by agarose gel electrophoresis to confirm the presence of band(s) of the expected
size.
MATERIALS:
LB-Kanamycin small plates
Inoculating loop
Bunsen burner
37°C incubator
Sterile culture tubes containing 3 mL LB-Kanamycin broth
Inoculating loop
Bunsen burner
Permanent marker
37°C shaking incubator
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2.0-mL sterile microfuge tubes
Sterile blue and yellow tips
Micropipettes
QIAprep miniprep kit
Nanodrop
10x EcoRI-HF Ready Mix
Sterile DI water
Ice bath in ice bucket
37°C heat block
5x loading dye
PROCEDURE:
A. Three to Four Days Before Lab:
1. Observe the plates inoculated from Part 2. Record the approximate number of colonies
on each plate. Make observations on the positive and negative controls, even if your
group did not prepare them.
2. Obtain 6 small LB + Kanamycin plates.
3. Label the bottom of each plate with your initials and a number beginning with 1.
4. For each plate, select a colony from your transformation plate that is separate from
other colonies. Using a sterile inoculating loop, quadrant streak the colony onto the
plate.
5. Wrap the plates in parafilm, invert and place in the 37°C incubator overnight.
B. One Day Before Lab:
1. Obtain 6 culture tubes containing 3 mL LB-kanamycin broth.
2. Label the tubes with your initials and the numbers that correspond to the plates that
you quadrant streaked.
3. Using a sterile inoculating loop, inoculate each tube with a different colony.
4. Grow the cultures in a 37°C shaking incubator for 16 hours. Growth for longer than 24
hours is not recommended since cells begin to lyse and plasmid yields may be reduced.
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C. Plasmid Prep
1. For each culture grown, label two sterile 2-mL microcentrifuge tubes with your initials
and the colony number.
2. Carefully decant (pour) each culture into the corresponding microcentrifuge tubes.
a. Do not cross-contaminate the samples.
b. Discard the culture tubes in the biohazard waste.
3. Centrifuge the cell suspensions at 10,000 rpm for 2 minutes at room temperature.
4. Pour off the supernatant into a waste container to be bleached.
5. Ensure that the RNase A has been added to buffer P1.
6. Resuspend the pelleted bacterial cells.
a. Work with one pair of microfuge tubes containing cells from the same colony.
b. Add 250 μL Buffer P1 to one of the tubes.
c. Resuspend the cells by vortexing, and transfer them to the second tube.
d. Resuspend the pelleted cells in the second tube by vortexing until no cell clumps
remain.
7. Add 250 μl Buffer P2 and mix thoroughly by gently inverting the tube 4–6 times.
a. Do not vortex, as this will result in shearing of genomic DNA.
b. If necessary, continue inverting the tube until the solution becomes viscous and
slightly clear.
c. Do not allow the lysis reaction to proceed for more than 5 min.
8. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6
times. To avoid localized precipitation, mix the solution thoroughly, immediately after
addition of Buffer N3. The solution should become cloudy.
9. Centrifuge for 10 min at 13,000 rpm at room temperature in a table-top
microcentrifuge. A compact white pellet will form.
10. Label spin columns and collection tubes with the corresponding codes.
11. Pipette the supernatants from step 14 to the corresponding labeled QIAprep spin
columns. Avoid taking the pellet.
12. Centrifuge at 13,000 rpm for 60 seconds.
13. Discard the flow-through into a waste container.
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14. Check that ethanol has been added to Buffer PE.
15. Add 750 μL Buffer PE to the column. Do not touch the pipette tip to the column
membrane.
16. Centrifuge at 13,000 rpm for 60 seconds.
17. Discard the flow-through into a waste container.
18. Centrifuge for an additional 2 min to remove residual wash buffer.
19. Label the sides of microcentrifuge tubes with the sample ID codes. Do not label the caps
in case they should break off in the subsequent centrifugation step.
20. Place the QIAprep column in the corresponding 1.5 ml microcentrifuge tube.
21. Add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each QIAprep spin column,
and let stand for 1 min.
22. Centrifuge at 13,000 rpm for 1 min. Place the lid on the rotor to prevent the microfuge
caps from breaking off.
23. Discard the QIAprep spin columns in the trash.
24. Use the Nanodrop to check the concentration of each sample.
a. Record the DNA concentration, 260/280 ratio, and 260/230 ratio.
b. The 260/280 and 260/230 ratios indicate DNA purity.
c. For pure DNA, the 260/280 ratio should be between 1.7 and 1.8. A ratio lower
than 1.7 indicates protein contamination. A ratio higher than 1.8 indicates RNA
contamination.
d. The 260/230 ratio indicates the presence of organic contaminants. Expected
260/230 ratios are between 2.0 and 2.2. If the ratio is lower, it may indicate the
presence of contaminants that absorb at 230 nm.
25. Use the DNA concentration of each sample to calculate the total amount of DNA
contained in 5 μL of sample. This is the volume to be used for the restriction digest
analysis.
a. A minimum of 20 ng of DNA is required for visualizing a band on a gel.
b. To visualize a fragment that is 1/10 the size of the initial plasmid, a minimum of
200 ng (0.2 μg) is required for the digest.
c. Too much DNA can lead to incomplete digest and/or smearing on the gel.
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d. Consult with your instructor to determine if the volumes of samples and
reagents need to be adjusted before making up your restriction digest master
mix.
D. EcoRI Restriction Digest
1. Prepare an ice bath.
2. Label 1.5-mL microcentrifuge tubes to correspond to the sample ID numbers and place
in the ice bath.
3. Add 13 μL sterile DI water to the bottom of each tube.
4. Add 5 μL of the purified plasmid to the corresponding tube. Pipette up and down gently
to mix.
5. Add 2 μL 10x EcoRI-HF Ready Mix to each tube. Add the enzyme directly to the solution
in the tubes and change tips between each sample. Mix by gently flicking the tube. The
EcoRI-HF Ready Mix already contains the buffer and loading dye, so no additional
components need to be added to the solution before loading on a gel.
6. Incubate samples at 37°C for 45 minutes.
7. Store at -20°C until the next class period.
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CULTIVATION INDEPENDENT ANALYSIS OF SOIL BACTERIA PART 4
In this lab, the digests from part 3 will be subjected to electrophoresis to confirm that the
plasmid had insert. The plasmid will then be prepared to send to the Delaware Biotechnology
Institute for sequencing.
MATERIALS:
Agarose
50x Tris Acetate EDTA (TAE) Buffer
SYBR Safe
250-mL Erlenmeyer flask
25- or 50-mL Erlenmeyer flask
Permanent marker
Gel electrophoresis chamber and set up
Blue and yellow tips
Micropipettes
15-mL graduated cylinder
1000-mL graduated cylinder
5x Loading Dye/Buffer
GelPilot Wide Range Ladder
PCR tubes
Sterile DI water
PROCEDURE:
A. Agarose Gel Electrophoresis
1. Each group will make their own gel using a 20 well comb.
2. Make a 0.8% agarose gel as directed in Part 2 of the soil microbe cloning lab.
3. For each plasmid isolated, transfer the volume used for the digests in part 3 to a new
PCR tube. Add an appropriate amount of 5x loading dye.
4. Load 8 µL of GelPilot Wide Range ladder to the first lane on the left. Then load each
sample in pairs, with the undigested in a well next to the digested sample.
5. Take a photograph.
6. Analyze the size of the fragments and determine if they add up to the size of the
amplicon that was used for cloning.
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B. Preparation for Sequencing
1. For every sample that has a cloned insert, obtain a PCR tube.
2. Write the corresponding code on the side of the PCR tube, neat enough for someone
else to read it.
3. For each sample, you need to submit 12 µL of plasmid at a concentration of 50 ng/µL.
a. Use the concentration of the sample obtained from the Nanodrop.
b. Use the equation C1V1 = C2V2 to determine the volume of plasmid required.
c. Add the calculated volume to the PCR tube.
d. Add enough sterile DI water to bring the volume to 12 µL.
e. Place your PCR tubes in the provided rack.
f. Place the remaining plasmid back in the freezer until you obtain useable
sequences.
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USING MEGA5 TO ANALYZE SEQUENCES
1.
MEGA5 is loaded on the computers in E225, E213 and E207. Or you can load MEGA 5.1 on
your personal computer. http://www.megasoftware.net/ It is free but you will need to
register.
2. Open MEGA 5.05 and in the drop-down menu under Align, select Edit/View Sequencer
Files. Select the forward sequence file (.ab1) that you wish to view. Repeat this step with
the matching reverse sequence file.
3. Make sure that the chromatogram looks clean without overlapping peaks as shown on
the left. If the chromatogram appears similar to the one on the right, discuss it with your
instructor.
4. Within the forward and reverse TraceEditor windows, search for the region of the 27F
sequence that is after the M (TGGCTCAG).
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5. Select the G at the 3’ end. Then click on the icon for masking upstream of the cursor.
6. Scroll to the right in the TraceEditor window, until the chromatogram begins to look
rough (usually around 800 bp). Select one of the bases in that region. Then click on the
icon for mask downstream from the cursor.
7. After the 5’ and 3’ regions are masked, select “Add to Alignment Explorer.”
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8. Click “OK” in the pop-up window.
9. Repeat the previous steps for the other sequence with the following modification.
Search for the sequence of the 1492R primer (GGTTACCTTGTTACGACTT). Mask
upstream of the 3’ T. Scroll in the window to the right to locate the region in which to
mask downstream. In the drop-down Edit menu, select “Reverse Complement” prior to
adding to Alignment Explorer. For aligning the sequences, the two reads must be in the
same direction.
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10. Since the two sequences only overlap by 100-200 bases, MEGA will not align them
properly. This must be done manually. The 5’ end of the reversed sequence will align
with a region towards the 3’ end of the forward sequence. You need to locate where the
overlap occurs.
In the Alignment Explorer window, select and copy between 10 and 15 bases at the 5’
end of the bottom sequence. In the pull down window under Search, select Find Motif.
Paste the sequence in the Find Motif window and click OK.
11. The motifs will be highlighted in yellow. Scroll across to locate the motif in the upper
sequence. Scroll back to the beginning and select the first base of the bottom sequence.
Use the space bar to move the bottom sequence to the right until the yellow regions
line up.
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12. When the sequences are aligned, the boxes above the sequences will be filled with a
symbol. Scan across the overlapping sequences to look for any mismatches between the
two sequences.
13. If any bases mismatch, look at the two TraceEditor windows to determine the correct
base. In the example below, the forward sequence has only one G and the reverse
sequence has two Gs. By looking at the TraceEditor windows, it can be clearly seen that
there are two Gs present in the reverse sequence. Therefore, another G is inserted into
the forward sequence.
14. Select the entire region of the reverse sequence that does not overlap with the forward
sequence. Select the first base on the bottom sequence that does not overlap. Then
hold down the Shift button and click on End. This will select the rest of the sequence.
Copy and paste it at the end of the forward sequence so that the forward sequence is
now a composite of the forward and reverse reads. Then delete the bottom sequence
from the Alignment Explorer.
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15. Export the file as a FASTA file. The file name should contain the soil code and sample ID.
In the example below, the file name is MNF003_03. MNF003 is the soil code, and 03 is
the sample code.
16. Return to the Alignment Explorer window. Select your sequence and click on the
colorful heart-shaped icon. A web browser will open to NCBI/BLAST and your sequence
will be pasted into the box for query sequence.
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17.Click the button next to Others under
Database and optimize for Highly
similar sequences. Click the BLAST
button and wait patiently for the hits
to be returned.
18. Look at the first hit to make sure that Strand=Plus/Plus. If Strand=Plus/Minus then you
need to return to Alignment Explorer. Under Data, select Reverse Complement. Export
the sequence to a FASTA file and redo the BLAST. For all sequences to be put into the
phylogenetic tree, they need to be in the Plus direction.
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19. Click on the number in the Max score column next to the first hit in the list of sequences
producing significant alignments.
20. In the Soil Results Form, record the clone code and whether or not the forward and
reverse sequences look good. In the column headed Blast BP Match, record the base
pairs that match in the query sequence for the first hit. In the example given, the Query
match begins at bp 58 and ends at 1448. Therefore, 58-1448 will be entered in the table.
In the BLAST % Match column, enter the percentage that follows Identities (91% in the
example).
21. Confirm the match using Ribosomal Database Project
(http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp). Click on the Choose File
button, select the FASTA file, and then click on Submit. Click on “view selectable
matches.”
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22. In the Soil Results Form under the RDP Identity column, enter the name with the highest
percentage match, other than uncultured. In the next column, enter the RDP % Match.
23. Open Word and then open the FASTA file that was just created. After the >, replace the
name with the same code you used for the file, followed by the RDP Match name. In the
example above >MNF003_03_Rhizobiales would be the top line in the FASTA file. Save
the file, and click Yes in the dialog box that appears. DO NOT SAVE the file as a Word
document. Programs that require FASTA files will not recognize Word documents.
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IDENTIFYING CHIMERAS FROM CLONES
1. In the drop down menu under Align, select Edit/Build Alignment. Select the button next
to Create a new alignment. Select DNA in the popup window.
2. In the Alignment Explorer window, choose Insert Sequence from File from the drop
down menu under Edit. Select the FASTA files containing the sequences you wish to
check.
3. After the sequences are all entered into Alignment Explorer, choose Export Alignment –
FASTA format in the dropdown menu under Data.
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4. Go to the Decipher website: http://decipher.cee.wisc.edu/FindChimerasOutputs.html
Enter in your email address, choose full-length sequences and browse to find the FASTA
file that you created in the previous step. Submit your request.
5. The file that is returned will include information about each sequence including the
identity of the best match, whether it is a chimera, and which genera make up chimeras.
Remove chimeras from further analysis including phylogenetic trees.
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