Bacterial ID Lab

 

 

In this virtual lab I assumed the role of a lab technician in a modern molecular biology laboratory.  I was responsible for providing lab results to medical doctors for use in diagnosing their patients. 

 

 

 

1. As the medical technician in charge of this investigation, what are you trying to determine about the tissue sample provided to you?

 

I am trying to identify different types of bacteria based in DNA sequences. My job is to identify a bacterial sample that is provided to me from a clinician.

 

 

 

 

 

 

 

2. How did you prepare the DNA to be used in this investigation?

• Put gloves on (so I didn't contaminate the sample)
• Got a wire loop and got a sample of bacterial colony from the culture dish
• Placed the bacterial colony sample into a microcentrifuge tube
• Used a micropipetter to add a digestive enzyme to the sample in the microcentrifuge tube.
• Disposed of the tip of the micropipette
• The enzyme was added to digest the cell wall so that the DNA sample could be obtained
• Let the sample sit for 3 hours
• Heated the sample to get rid of the enzymes
• Placed microcentrifuge tube into a hot bath to be heated at 100C
• Placed microcentrifuge tube into the centrifuge
• Added a counter balance
• Collected the DNA from the liquid part of the sample, the supernatant, placed it into the PCR tube 

 

 

 

 

 

 

 

3. Describe how PCR is used to make copies of DNA sequences. Use the animation and notebook entries in the PCR Amplification step to guide your answer. Note that you may replay the animation as needed.

The DNA sample that was obtained in the sample prep stage is placed into the PCR tube. Then the PCR master mix is added to the sample in the PCR tube. Next, the PCR tubes are placed into the PCR machine to begin the DNA replication process. The doubloe stranded DNA is heated to 95C by the PCR machine for 30 seconds. This step seperated the 2 strands of DNA. Then the PCR machine cools to 60C for 30 seconds so that the primers can bond. or anneal, to the single DNA strands. Next, the PCR machine heats up to 73C for 45 seconds for the extend step to heppen. In this step the polymerase is used to extend the copy or the DNA. This process is then repeated 30 or more times to get multiple copies of the DNA sequences.

 

 

 

 

 

 

 

 

 

4. Summarize the technique used to purify the PCR product.

 

1.  Get a collection tube and place a microconcentrator column into it
2. Place 400 microliters of buffer sloution and all of the PCR product into the column
3. Spin the column in the centrifuge at 3,000 revolution per minute for 15 minutes to seperate the PCR into the column
4. Invert the column into a new collection tube and place 50 microliters of the buffere into the inverted column
5. Place the column into the centrifuge and spin at 3,000 revolutions per minute for 2 minutes to loosen the DNA into the collection tube
6. Dispose of the microconcentrator column

 

 

 

 

 

 

5. What is produced during the sequencing prep PCR run? Use the animation and notebook as needed in thinking through your answer.

 

 The sequencing prep PCR run produces many DNA copies at different lengths. The primer bonds to the strand and copies the sequences of the DNA, but terminates at different lengths with a fluorescent marker.

 

 

 

 

 

 

6. Describe how the automatic sequencer determines the sequences of the PCR products.

 

 

 The automatic sequencer performs the process of gel electrophoresis on each of the tubes of DNA. The gel electrophoresis seperates the DNA molecules by the different sizes. The automatic sequencer uses capillary tubes that have one end attached to a syringe with a buffer solution in it. The capillary tube is filled with the buffer solution and the end opposite of the syringe is placed into one of the tubes that hold the DNA. The machine then applies an electrical current to the tube. The end of the tube that has the syringe has a positive charge and the end that is in the DNA has a negative charge. The DNA has a negative charge, so when it comes in contact with the negative current, the molecules travel towards the positively charged end, the smallest parts moving quicker than the bigger parts. The tube moves through a laser beam. The beam causes the fluorescent marker to become excited and then the optical detectors can detect their color.

 

 

 

 

 

 

 

 

7. What does BLAST stand for?

 BLAST stands for Basic Local Alignment Search Tool.

 

 

 

8. What conclusions did you make using the results of the BLAST search? Did these conclusions support a clinical diagnosis for the patient (what disease did they have)?

I identified the bacteria as being "Bartonella Henselae". This patient would have Cat Scratch Disease (CSD) and would have contracted the disease from cats. Cat Scratch Disease has symptoms of swelling of lymph glands, and possible skin lesions, fever and fatigue.