Microbial Diversity Homepage

Microbial Biodiversity Project Protocol

The following represents a summary of the progressive steps that were used in a group research project on the  microbial biodiversity found in ponds and streams in the Princeton, New Jersey area.

The value of science is the process as well as the product. There are many occasions where unanticipated results were obtained.  Our results allowed us to re-evaluate our protocols and revise the experimental design.   Upon trouble shooting, we were able to determine that our universal primer was incorrect.  So, if at first you don't succeed, try, try again! 

Objective:

Our objective was to sample the microbial biodiversity in stream and pond water in the Princeton, New Jersey area.  After determining the microbial biodiversity, this information was used to develop dendrograms and unrooted trees.  The graphics allowed us to consider the potential evolutionary  relationships among the microbes surveyed.

Our genetic information was based on extracting, amplifying and sequencing a highly conserved region of the bacterial genome - the 16S region which codes for ribosomal DNA. 

Overview of the Steps Taken in This Research Project:

A. Collect and extract DNA from microbes recovered from filtration

B. PCR (Polymerase Chain Reaction) DNA to isolate and amplify the specific gene on the bacterial DNA (our 16S ribosomal insert is 1.5kb)

C. Run our PCR product on electrophoresis gels. If we get DNA product, clone and grow on agar plates.  If we don't get DNA product, go back to PCR.

D. Colony Cloning Reaction - to separate and isolate taxa

E. PCR colonies of plasmid template (M13 forward primer)

F. Run PCR products on gels.  If we get DNA product, then we will sequence.  If we don't get DNA product, we go back to cloning process.

G. Sequence with cycle sequencer and read sequence with BLAST Program to determine microbial identity.  Finally, we align the sequences using PHYLIP to determine the phylogeny of the bacteria to understand evolutionary relationships.

A. Collecting, Filtering, and Extraction Procedure  (top)

Collection: We collected a liter of water from the pond at Lawrenceville School and a liter of water from a local stream using sterile plastic bottles.  (The water sample should be collected from the area below the surface and above the sediment.)    Filtering: Each sample was vacuum filtered through 1.2 um Nucleopore polycarbonate filters (Millipore) and then through 0.22 um Duropore membrane filters (Millipore) to remove organisms other than bacteria.  The filters may be stored at -4oC until processed.

Extraction of DNA

Materials:

• DNA extraction buffer = 100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate buffer [pH 8.0], 1.5 M NaCl
• Proteinase K (10 mg/ml)
• 20% SDS
• Chloroform-isoamyl alcohol (24:1 v/v)
• 70% ethanol
• sodium phosphate buffer [pH 7.0]
• TE buffer [pH 8.0] = 10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH 8.0]

Procedure:

1. Cut filters into small pieces with sterile scissors (EtOH flamed) and put into a 40 ml centrifuge tube (Teflon, Fisher).  Add 13.5 ml DNA extraction buffer and 100ul proteinase K (final concentration 10 mg/ml, dissolved in DNA extraction buffer).  Mix well (if using 40ml Oakridge tubes, use 10 ml DNA extraction buffer and 67 ul proteinase K so that final extract with chloroform-isoamyl alcohol will fit in one tube).

2. Put the tube in a 37^oC shaking incubator and shake at 225 rpm for 30 mins.

3. Add 1.5 ml 20% SDS solution to the sample and incubate at 65^oC for 2 hrs.  Gently end-to-end invert every 15-20 mins.

4. Centrifuge the sample at 6,000 x g for 10 min at room temp. and transfer the supernatant to a new tube.

5. Resuspend pellet in 4.5 ml DNA extraction buffer and 0.5 ml 20% SDS.  Incubate at 65^oC for10 min.

6. Centrifuge the suspension at 6,000 x g for 10 min at room temp. and collect the supernatant.

7. Repeat the treatment of pellet one more time.

8. Pool the supernatant and measure the volume, then add equal volume of chloroform:isoamyl alcohol (24:1); mix well.

9. Centrifuge the mixture at 6,000 x g for 10 min, and collect the supernatant, then add 0.6 volume of isopropanol to the supernatant, let DNA precipitate at room temp. for 1 hr (or overnight ).

10. Centrifuge the mixture at 15,000 x g for 30 min. at room temp and collect the pellet.

11. Add 2 ml 70% ethanol to wash the DNA extract (pellet), centrifuge again and remove the ethanol.

12. Let DNA dry in the laminar hood, then add 500 ul autoclaved TE buffer to dissolve DNA extract, and store at -4^oC for further processing. 

* If humic content is high (i.e., extraction is dark, like tea), use a Sephadex Purification.

B. PCR Procedure:  (top)

Strict aseptic procedures are essential to obtaining pure microbial DNA.  Bacteria is everywhere, so sterile lab procedures are essential. 

Eppendorf tubes were subjected to UV light to cause any ancillary DNA (from surrounding bacteria) to break apart.  Clean pipette tips were used with the exchange of all liquids. Latex gloves were worn during all procedures.

At this point, half of the reactions were carried out using  PCR beads (bought from a kit); the other half used our master TAq mix.

*All measurements are in microliters

When all materials are mixed, place PCR reactions in the thermal cycler which was run through 30 sequences.

Thermal Cycler parameters:

Insert at this point should have been amplified and then run through gel electrophoresis

C.  Electrophoresis Gel:  (top)

Results were obtained from the tubes that were treated with the master TAq mix; no results were obtained from the tubes that contained the PCR beads.

Results were obtained from stream DNA but not for soil or pond microbes.

D. Colony Cloning Procedure (Kit from Invitrogen)  (top)

TOPO Cloning Reaction: 

Reagents added to chemically competent E. coli Fresh PCR product                            4 microliters Salt solution                                       1 microliter TOPO vector (plasmid)                     1 microliter Final Volume                                     6 microliters

*Chemically competent cells have weakened cell membranes via treatment with Ca₃(PO₄)₂

*The plasmid vector is supplied linearized with single 3'-thymidine (T) overhangs for TA Cloning and Topoisomerase I covalently bound to the vector (referred to as "activated" vector)

1.  Mix gently and incubate for 30 minutes at room temperature (longer incubation time is needed because insert is long … 1.5 Kb)

2.  Place the reaction on ice to stop reaction

Prepare LB lacZ⁺ (genes that breaks down a sugar - blue component) Amp^R (Ampicillin resistance gene) media plates (from Invitrogen, imMedia Amp Blue packets)

(If our microbial colonies grow and turn blue, they have not accepted the insert into the plasmid and are unusable.  Our plasmid has a gene for ampicillin resistance,  so our colonies will  be able to grow normally on the plate . The ampicillin is included to eliminate foreign environmental microbes that could provide potential contaminate DNA.)

Four plates for each - soil, stream 1.2, stream 0.2, pond 1.2, control

One Shot Chemical Transformation Protocol

1.      Add 2 microliters of the TOPO cloning reaction (table above) to a vial of One Shot Chemically Competent E. coli and mix gently

2.      Place on ice for 5-10 minutes

3.      Heat shock the cell for 30 seconds at 42^oC without shaking

4.      Immediately transfer the tubes to ice

5.      Add 250 microliters of room temperature SOC medium

6.      Cap the tube tightly and shake the tube horizontally at 37^oC for 1 hour

7.      Spread 50-100 microliters from each transformation on a pre-warmed selective agar plate and incubate overnight at 37^o C

E.  PCR Colonies:  (top)

After 12 hours of incubation, bacterial colonies were “picked” with toothpicks. Colony PCR is done to amplify cloned insert.

1.We each picked five to ten colonies.

2.We picked a small amount of the colony because proteins and lipids could interfere with the reaction.

3. We transferred each colony to a separate tube with PCR reaction chemicals.

Colony PCR Master Mix:

Each 0.5 milliliter Eppendorf tube              Master Mix

1 microliter primer 1                                 45 microliters primer 1

1 microliter primer 2                                 45 microliters primer 2

23 microliters water                                  1035 microliters water

When all is mixed, place PCR reactions in the thermal cycler.

Thermal Cycler parameters:

Place PCR reaction material on electrophoresis gels.

F. Gel Prep  (top)

1. Tape ends of gel trays and place combs in trays 2. Pour 1x TBE buffer to help conduct electricity in the gel rig 3. DNA is negatively charged, so run from the black (-) lead to the red (+) lead 4. Mix 1% agarose gel mix and add 0.8 microliters of ethidium bromide 5. Pour 1% agarose/bromide mix into tray (allow to dry ... remove tape) 6. Mix 7 microliters of PCR product with dye 7. Put trays into the gel rig and load 8. Turn on gel rig.  Run gel for 15 minutes

* Our gels gave us inconclusive results.  

Due to our inconclusive results we decided to alter our PCR parameters.

1.   When collecting colonies, we used pipette tips instead of toothpicks.

2.   We picked new colonies from  the petri dishes.

3.   We left the tips in the PCR master mix solution for a couple of minutes to allow for more colonies    to enter into the master mix

4.   We changed  the PCR parameters by increasing both the time and temperatures

Thermal Cycler parameters:

5.      Run PCR with new colony picks

6.      Run electrophoresis gels with PCR products

*Gels again proved to be inconclusive as to whether we had DNA from the PCR.

Based on the fact that we had two inconclusive trials in trying to isolate microbial DNA samples, we held a group meeting to determine our next step. The following were discussed:

1.   Are our primers working?

2.   The agarose gel may not be appropriate for primers over 1,000 base pairs.

3.   Is boil preparation hot enough?  We need to get to 100⁰C  to lyse the cells and inactivate any enzymes that we don’t want.

4.   Are too many cells in the product inhibiting the PCR reactions?  (This could explain the material that remained in the wells after the electrophoresis gel was run the second time)

5.   "Colony PCR is sometimes more art than science."  Kurt Lienau

6.    We should use the control plasmid for comparison in the PCR kit because we are getting some unexplainable results from our standard.

7.    Should we run gels without putting dye in with our E-gels? (Perhaps the faint bands are being covered by the dye bands.)

We then loaded PCR samples from prior reactions and a standard on E-gels (without loading dye) and developed the gel for 15 minutes.

No results from gels were conclusive, except for a "weak" showing from stream 1.2.

We are going to start back at step B of our experiment to try to eliminate any prior experimental error.

We poured agar plates.  Four plates were each used for Stream 1.2, Stream 0.2, Pond1.2.

Original  PCR products were then used for colony cloning reactions.

1.  Mix gently and incubate for 30 minutes at room temperature (longer incubation time is again used because the insert is long … 1.5 Kb)

2.  Place the reaction on ice to stop reaction

It was discovered that the inconclusive results were a result of using a primer that was incompatible to our studied gene region . The primer we were using was mislabeled by the company from which it was purchased  new M13 Universal Primer was acquired. 

One Shot Chemical Transformation Protocol:

1.   Add 2 microliters of the TOPO cloning reaction (table above) to a vial of One Shot Chemically Competent E. coli and mix gently.

2.    Place on ice for 5-10 minutes

3.    Heat shock the cell for 30 seconds at 42^oC without shaking

4.    Immediately transfer the tubes to ice

5.    Add 250 microliters of room temperature SOC medium

6.    Cap the tube tightly and shake the tube horizontally at 37^oC for 1 hour

7.    Spread 75 microliters from each transformation on a prewarmed selective plate and incubate overnight at 37^oC

Agar plates were incubated overnight. Colonies grew on Stream 0.2 and Pond 1.2.  plates. Our Stream 1.2 plates did not grow any colonies.  This may be a result of our plates being turned over when they were still wet.

Boil Prep and PCR Protocol:

1. UV irradiate 40  0.5 milliliter Eppendorf tubes.

2.  Place 100 microliters of buffer solution (TE - Tris EDTA) in each tube.

3.  Use a pipette tip to collect the colonies and place in each tube.  (Ten from the Stream 0.2 and thirty from the Pond 1.2)

4.  Allow tips to sit momentarily in the buffer solution to slough off a few cells. (We are trying to correct error of collecting too many cells that might have stayed in the gel well)

5.  After colony collection, allow solution with colonies to sit for 10 minutes in 100⁰C boil prep (to break open the bacterial cells).

6.  Place 2 microliters  of each sample from the buffer/colony solution into 0.2 milliliter PCR tubes with bead.  Add 22 microliters of water, and 0.5 microliters of each primer into the tubes. PCR colonies.

7.  Thermal Cycler parameters:

8. PCR products were then put through gel electrophoresis.

9. Favorable results were finally obtained!! Thirty-four out of forty samples were usable for cycle sequencing.

            Stream and Pond                                                Pond and Pond

The PCR products were then gene cleaned through a QIAquick PCR Purification Kit and readied for the cycle sequencer. The cycle sequencer gave us a genetic base sequence for each unknown microbe. This information was put though the BLAST program in an attempt to determine the identity of the microbes.

QIA Quick PCR Purification Protocol:  This protocol is designed to purify single or double-stranded DNA fragments form PCR and other enzymatic reactions.  Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.

*Add ethanol (96 -100%) to Buffer PE before use

1.      Add 100 microliters of Buffer PB to 20 microliters of the PCR sample and mix. 

2.      Place the QIAquick spin column in a provided 2 ml collection tube.

3.      To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 seconds.

4.      Discard flow-through.  Place the QIAquick column back into the same tube.

5.      To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30-60 seconds.

6.      Discard flow-through and place the QIAquick column back in the same tube.  Centrifuge the column for an additional 1 min at maximum speed.

7.      Place QIAquick column in a clean 1.5 ml  microcentrifuge tube.

8.      To elute (bring back into solution)  DNA, add 50 microliter Buffer EB or water to the center of the QIAquick membrane and centrifuge the column for 1 min.  Alternatively, for increased DNA concentration, add 30 microliters elution buffer to the center of the QIAquick membrane, let the column stand for 1 minute and then centrifuge.

9. Materials are now ready for cycle sequencing master mix.

Protocol for Cycle Sequencing Master Mix:

Big Dye            2 microliters                                        

EXT                2 microliters

Primer              0.5 microliters

Template          1.0 microliters

Water               4.5 microliters

Add 9 microliters of Master Mix to each 0.2 strip cap Eppendorf tube and add 1 microliter of template.  DNA samples were then cycle sequenced.   

An Isopropanol Precipitation reaction is done to clean up our DNA cycle sequencing reaction, removing any unwanted materials.

Isopropanol Precipitation Protocol:

1.    2.5 volumes (25 microliters) IPA to your 10 microliters of CSR product

2.    Spin for 1/2 hour

3.     Draw off 25 microliters of supernatent , being careful to not draw off  the DNA pellet

4.     Repeat with the 25 microliters of IPA and shake; spin down

5.     Draw off 25 microliters of supernatent and allow to air dry

6.    Resuspend in loading buffer

7.    Add 2.5 microliters of formamide dye  (loading gel dye + formamide)

8.    Heat to 95^oc for 1 min. and then cold shock

9.    DNA is now ready to load on a polyacrylamide gel to determine the sequences of our inserts.

G. Sequences from gel with corresponding BLAST Results:  (top)

Table 1:  Blast Results for Stream and Pond Bacteria Sequences

Back to Home Page