BISC209/S12: Lab5

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Wellesley College-BISC 209 Microbiology -Spring 2012


LAB 5: Soil Community Bacterial Member Identification by 16s rRNA gene sequencing

By this week we hope that you have pure cultures from your enrichment/selection and isolation work. If you have isolated, pure colonies on your isolation streak plates, congratulations!. Make sure that these potentially pure cultures have colonies that look like the original source colony and that all of the colonies look the same (they can be of different size). If you are having trouble obtaining a pure culture, consult with your instructor BEFORE LAB 5. We can't identify your isolate by 16s rRNA gene sequencing if it is not a pure culture.


Today you will begin the identification of culturable bacterial isolates in your soil habitat. Using your culture isolates as the template, you will amplify, in a polymerase chain reaction (pcr), part of the 16S rRNA gene from your pure cultures of bacteria using "universal" bacterial primers and a proof-reading DNA polymerase. If you are unfamiliar with the basic theory behind pcr amplification of target DNA, please review it in at the following web site: PCR animation



Part A: Prepare Lysates from pure cultures of Bacteria of Interest



1. Each student will obtain DNA from 4-7 unique bacteria (up to 24 per team). Use well isolated colonies from your best plate (one that's unquestionably a pure culture) for each of your bacteria of interest. Consult with your partners to make sure that your team is identifying bacteria that are unlikely to be of the same species and NOT likely to be mold or nonbacterial microbes.

2. Each students needs pcr tubes of your team color (0.2ml vol.). You will need one for each of your isolates. Wear new gloves when you take them out of the jar on the instructor bench and label each with a unique code to indicate each isolate you want to id. DO NOT CONTAMINATE THE INSIDE OF THE CAP with your skin flora. Use your sampling habitat id (site A, B, C, or D) and a unique set of numbers that your lab instructor will assign each student. Numbers will be found on the board.


4. Touch a well isolated colony from a pure culture with a sterile P10 micropipet tip (the tiny ones, not the P20 tips-use the tip as if it were a toothpick) and resuspend a barely visible amount of bacteria adhering to the tip by swishing the tip around in the appropriately labeled tube with water and detergent. Resist the urge to pick up too much cell material, but be aware that some of your isolates, particularly those dry, powdery Actinomycetes (like Steptomyces) are hard to get any cells to adhere. For those, it is ok to take a bit more. The tinest bit is enough, but make sure there is some part of the colony going into the lysate. Putting in too many cells can inhibit the pcr reaction and be as bad as putting in none.

5. Repeat for your other isolates-- into separate tubes.

6. Boil all samples for 5 minutes. This will lyse the cells and inactivate bacterial enzymes. You can boil in a heat block or in the thermal cycler-- if your instructor setup a program to boil and you used the small pcr tubes. Since she did and you did, we will boil the tubes in the thermal cycler today. Be careful when you remove the tubes from the thermal cycler. Point them away from you and ease the lids open while still covering the outside of the tops with your gloved fingers. Do this slowly and carefully with the opening pointed away from you. You don't want the caps to pop and make an aerosol of your bacteria and you don't want to lose your lysate.

Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above

Note: We are using a kit from New England Biolabs called Phusion® High Fidelity PCR Master Mix with HF Buffer | Because you are using a kit to amplify the bacterial 16s rRNA gene and the manufacturer does not have to disclose how to make each reagent from scratch, you can not include typical instructions for making stock reagents (ingredients and concentrations)in your Materials & Methods section. However, since the manufacturer's website includes instructions for how to use this product, you can refer your reader to that information and explain simply that you used this kit to accomplish the gene amplification according to manufacturer instructions. Keep in mind that you WILL have to give specific information about the gene template, the primers (DNA sequences), and the thermal cycler program since those things are not universal but specific to the primers and DNA we want to copy. (Size and GC content are crucial variables). "Universal" Bacterial Primer sequences for the 16s rRNA gene are 1492R reverse primer TACGG(C/T)TACCTTGTTACGACTT and the forward primer 27F is AGAGTTTGATC(C/A)TGGCTCAG. Note that the () indicates where either of two bases can be used. Our primers are purchased from Integrated DNA Technologies (IDT) at |

Setting up the PCR Mix

Using a P2 or P10 and filter tips (remember that the P2 has two red decimal place volume indicators while the P10 only has 1 red decimal place indicator. MAKE SURE YOU HAVE DIALED IN THE CORRECT VOLUME!), add 4 microliters of your boiled lysate (containing the template DNA) to the prealiquoted 46 microliters of master mix (contains DNA polymerase, dNPTs, MgCl2, and buffers), primers and nuclease free water mixture described above (for a total volume of 50 μL) in clearly labeled pcr tubes of your team color. Make sure you label on both the top and sides of the tube. The tubes are tiny (holds only 0.2ml vol) and it is hard to write on them legibly but doing so is very important. You will have to use a unique identification code for your isolates and keep the key to the code in your lab notebook (also give a copy to your instructor).

Component TABLE

Component amt. in a 50 μl
Final Conc.
DNAase free
16 μl
2x Phusion Master Mix 25 μl 1x
27F primer 2.5 μl 0.5 μMolar
1492R primer 2.5 μl 0.5 μMolar
template DNA 4 μl 50-250ng
optimum is 100ng of DNA/reaction


  • 27F AGA GTT TGA TCC TGG CTC AG Universal Lane et al. 1991

  • 1492R (s) ACG GCT ACC TTG TTA CGA CTT Universal Lane et al. 1991 =





Thermal Cycler Program:
3 step program

Cycle Step Temperature Time # of Cycles
Initial Denaturation 98C 30 sec. 1
10 sec
30 sec
30 sec
Final Extension 72C
10 min

The pcr will run for 45min or so.

Part C: Agarose Gel Electrophoresis of Clean PCR PRODUCT





Procedure for Agarose Gel Electrophoresis of PCR products
You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 5 microliters of well mixed loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix by pipetting up and down before loading all 10 microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer with Sybr Safe DNA stain. Sybr Safe is a proprietary reagent from Invitrogen that cross-links a fluorescent compound to DNA and allows DNA to be visualized under UV light. It is prepared and used according to manufacturer's directions Apply your samples to the wells that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.




Make sure each of your pcr products is clearly labeled with your initials, lab section (Tues or Wed), soil sample identifier code letter, your unique code information for each bacterial isolate and the conc. of the DNA in ng/μL.


Part D: PCR Purification (Clean-Up)

It is important that your pcr product be free of left over DNA polymerase, primers, and free nucleotides before a sequencing reaction is attempted on it. Your instructor will let you know if you will do this purification today after you have loaded part of your sample on the gel or if she will do it for you later, or, perhaps, we might have the sequencing facility do it for us. The important thing for you to know is that it must be done and that there are several methods commonly used to accomplish this purification. One of the simplest is an enzymatic teatment. If we do this clean=up in house, we will use an Exosap-it® kit from USB.

USB ExoSap-iT PCR Product Cleanup (Product Number 78200)

1. Remove ExoSAP-IT reagent from -20C freezer and keep it on ice throughout this procedure.
2. Mix 5μL of a post-PCR product with 2μL of ExoSAP-IT reagent for a combined 7μL reaction volume. NOTE: When treating PCR product volumes greater than 5μL, simply increase the amount of ExoSAP-IT reagent proportionally.
3. Incubate at 37C for 15 minutes to degrade remaining primers and nucleotides.
4. Incubate at 80C for 15 min. to inactivate ExoSAP-IT reagent.
6. Treated PCR products should be stored at -20C until required.


Part E: Submitting Amplicons for Identification

Before your next lab, your instructors will prepare a 96 well plate of your pcr products that showed successful amplification of the 16s rRNA gene. We will send these samples to a commercial DNA sequencing facility that will "clean-up" our pcr products by removing primers, polymerase and unused dNPTs that might inhibit the reaction. They will optimize the pH and the concentration of the DNA for chain termination sequencing of your amplified 16s rDNA (16s rRNA genes). The sequencing reaction will be done for you and the resulting DNA fragments will be separated by size in an automatic sequencer with a laser that can read the fluorescent tags on the end of each fragment. This crucial part of identification of bacterial members of your soil community will be done for you and the DNA sequences will be reported back as .ab1 files that will require special software to analyze. We want you to understand exactly what's going on in those automatic sequencing machines. Make sure you understand how chain termination (Sanger) sequencing works. We should have the sequencing data back by the time we are scheduled to learn how to analyze the data so we can find out, we hope, the identity of a few members of the bacteria in your soil community.


Testing for Antibiotic Production

Start the Testing for Antibiotic Production.


(This testing will take 3 weeks.)
Week 1:








Characterization of Soil Bacteria: Maintaining Pure Cultures of Isolates


Preparation for Next Lab

Tests of your isolates will continue next week. You will use the sub-cultures made today or their antecedents or descendants. Depending on the test, you may need a fresh liquid broth culture, or an isolation streak plate culture on NA. You will need to plan ahead to prepare the appropriate cultures so that they will be ready to use when needed. The number of hours it takes from inoculation until a bacterial culture moves from log to stationary or death phase depends on its generation time, the concentration of the inoculum, and other factors. If you have a reasonably fast growing bacterial strain, you should make a subculture into Nutrient broth solid and liquid medium about 24-48 hours before you are to set up a new test. If you have a particularly slow grower, those cultures need to be set up earlier than that. Keep track of how fast each of your soil bacterial isolates grows and plan accordingly.

Reference Information

Link to the electronic edition of | Bergey's Manualsthrough Springer ebooks.
Use these reference resources to decipher the meaning of the results of the morphologic, metabolic, physical and other tests you will perform or to look up the usual characteristics of the isolates that are identified by 16s rRNA gene sequencing.













12. Wash your hands.


M&M: Compose a draft of your Materials and Methods section of your final paper with the following general sections:
1) Obtaining a soil sample & making a soil extract 2) Enummeration of microbes in a soil community;
    a) Cuture-Dependent Colony Count
    b) Culture-Independent Direct Count
3) Identification of bacteria in a soil community by 16S rRNA gene sequencing;
    a) Isolating Bacteria of Selected Groups to Pure Culture
    b) Amplifying the 16srRNA gene by colony pcr & preparation for Gene Sequencing
4) Soil Microbial Co-operation & Competition:
    a) Culture-Dependent Microbial Community Assessment of Functional Metabolic Diversity
       1) Carbon source utilization pattern and diversity
    b) Co-operation and Competition among Cultured Bacterial isolates from a Soil Community
        1) Prevalence of Starch, Cellulose, and Phosphate digesters or processors

In Lab 6-8, you will doing most of the assessment of your isolates' through a battery of tests and special stains, a few of which require some preparatory work. Make sure you set up a fresh broth culture of appropriate media for each isolate a few days before your next lab and have a fresh streak plate of each isolate ready for next week's tests. Familiarize yourself with the tests and stains you will perform. Make sure you have outlined the protocols in your lab notebook and started any necessary cultures on appropriate medium so that they will be ready to use in lab at the appropriate time:

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