Name:___________________________
Bacterial ID Lab at Howard Hughes Medical Institute
download the app.... or go to the following webpages
http://www.hhmi.org/biointeractive/vlabs/index.html
or
www.hhmi.org >> resources & publications >> students >> biointeractive >>
virtual labs > "The Bacterial Identification Lab"
The procedures necessary for extracting and isolating DNA of an unknown bacteria
are costly, time consuming, and can even be dangerous in that it can expose a lab
technician to pathogens. Fortunately, technology has given
us virtual labs that will show how these procedures are
done without the mess and the fuss. In this internet activity,
you will use a virtual lab.
Basic Steps:
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Prepare a sample from a patient and isolate whole
bacterial DNA.
Make many copies of the desired piece of DNA.
Sequence the DNA.
Analyze the sequence and identify the bacteria
Learning Objectives:
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What kind of patient samples are used for the purpose of identifying possible
pathogens?
What does PCR do, how does it work, and why is it useful?
How do you separate the desired DNA from all others?
How does an automatic DNA sequencer work?
Why is it possible to use a DNA sequence to identify bacteria?
Instructions: The virtual lab is divided into six sections that walk you through the
procedures involved. Don't forget to read the notebook section as well, which has
further explanation about what you are doing and why. As you complete each
section, answer the corresponding questions.
Intro: What piece of DNA is being used to identify bacteria?
Sample Prep
1. The first step in the extraction process uses a wire loop to do what?
2. Why are the digestive enzymes necessary for DNA extraction?
3. Why must you then head the micro-centrifuge tube?
4. The centrifuge separates the cellular content into two distinct layers. Describe
these layers and what is contained within them.
PCR Amplification
5. PCR stands for ________________________________
6. What is found in the PCR Master Mix?
7. What is a primer? (see link)
8. Describe what happens during the 3 PCR stages
Melt
Anneal
Extend
9. After 30 cycles, how many copies of the initial DNA strand have been
produced? Why is this called "amplification"?
PCR Purification
8. To confirm that the PCR worked, three lanes are run that contain 3 materials.
What materials are in the 3 lanes? Lane 1 __________________________
Lane 2 ______________________ Lane 3 _______________________
9. What is the overall purpose of the purification stage?
Sequencing Prep
10. How are dideoxynucleotides used in sequencing? (learn about cycle sequencing
link)
11. Why are multiple primers used?
12. Describe the “sequencing brew” that you added your purified PCR to.
11. The purpose of the second PCR is not to create identical copies like the first
PCR you ran. What is the purpose of this second PCR?
DNA Sequencing
12. What is the final PCR product, the stuff contained in your 12 tubes?
13. What is gel electrophoresis?
14. Why does DNA move through the capillary tube towards the syringe?
Sequence Analysis
14. What is BLAST? (see The science behind sequencing link)
15. What does it mean for a region of DNA to be “conserved?”
16. The higher the score the ______________ the match (see learn more about
BLAST link)
17. What does a low E value mean?
18. What is the identity of the bacteria in your sample? Follow the steps listed on
the page and be patient, statistically aligning the sequences can take time.
Sample A (lymph node) _______________________
Sample B (stool sample)_______________________
Sample C (urine sample) _______________________
Sample D (blood sample) _______________________
Sample E (sputum sample) _______________________
Sample F (stool sample) _______________________