BISC209: Lab7

ID Cultured Bacteria by 16srDNA Sequencing and Analysis

Besides working on getting your soil sample DNA isolated, amplified by pcr, inserted in a cloning vector, transformed into E. coli, and the 16s rDNA insert sequenced in an automatic sequencer so you can identify a more represenative scope of the bacterial flora in your soil sample, you have been working simultaneously, through traditional microbiological culturing techniques, to isolate and identify some of the culturable bacteria by morphology and metabolism differentiation. Look how much you have accomplished in these few short weeks!!

By this point you have isolated pure colonies of some soil bacteria on general and enrichment media and you have gotten some preliminary information about the morphologic and metabolic characteristic of the bacteria you have chosen to identify. You will continue learning about how these bacteria are different from one another and how they contribute to their community. At the same time we want to identify these bacteria by their 16s rDNA unique sequences in a similar way as we did for our unknown general soil sample bacteria. This time we will use a Taq polymerase that will not be as accurate as our proof reading polymerase we used previously, but should be good enough to get our identifications.

Identify Bacteria from DNA from Isolated Pure Colonies

Choose 4 different isolated bacterial colonies per person

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Links to Labs in the Soil Microbes Project

Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9

We would like to thank Charles Deutsch, Patricia M. Steubing; Stephen C. Wagner and Robert S. Stewart, Jr.; Kyle Seifert, Amy Fenster, Judith A. Dilts, and Louise Temple; and the instructors of the Microbial Diversity Course at the Marine Biological Lab in Woods Hole, MA for their valuable assistance in the development of these labs.

Lyse Cells by boiling
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PCR amplify 16srDNA with the bacterial primers: 8F and 1492R

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Visualize PCR product by agarose gel electrophoresis ⇓
Prepare PCR product for DNA sequencing
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Submit PCR products for DNA sequencing

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Determine id of cultured and isolated soil organisms with sequence comparisons to database

Agarose Gel Electrophoresis of PCR products

DNA is uniformly negatively charged and will,therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA.

You will run your pcr products on a 1.5% agarose gel in hopes of seeing what? How many fragments and of what size do you expect to see in a successful amplification of 16s rDNA using the 8F and 1492R primer pairs?

PCR CLEAN UP with EXOSAPit

We will have to clean up our reactions for sequencing to remove the excess dNTPS, primers, and pcr product contaminants using EXOSAP it.

When PCR amplification is complete, any unconsumed dNTPs and primers remaining in the PCR product mixture will interfere with these methods. ExoSAP-IT removes these contaminants. ExoSAP-IT contains two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, together in a proprietary buffer. It removes unwanted dNTPs and primers from PCR products. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. Shrimp Alkaline Phosphatase removes the remaining dNTPs from the PCR mixture.<BR

PROTOCOL for EXOSAPIT
For those pcr reactions that resulted in a single product of the expected size, combine 1.5 microliters of your pcr reaction with 3.5 microliters of EXOSAPIT master mix in clearly labeled pcr tube of your team color. Place those tubes in the thermal cycler and record the position of each of your samples on the 96 well sheet provided. For any amplifications that were not successful or resulted in multiple fragments of the wrong size, consult with your instructor about whether or not to include them.

The PCR program will be: 37C for 30 min. and 80C for 15 min. (to denature the enzymes). We will use 3 microliters of the ExoSapit reaction for sequencing.

Links to Labs in the Soil Microbes Project

Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9