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INTRODUCTION
Before starting this activity it is recommended that you read Polymerase Chain Reaction in your
textbook, pages 169-170.
Go to https://www.hhmi.org/biointeractive/explore-virtual-labs. Scroll down and click on “The
Bacterial Identification Virtual Lab.” Maximize the screen if you wish. Answer the following questions
in the spaces provided.
Click to Enter the Lab. (Click the window on the left-hand side of the screen to enter the lab.) As you enter
the lab, follow the instructions in the lab (left-hand window). Using the information in the Notebook
window on the right, answer the following questions.
What is "16S rDNA," and how is it used to identify species of bacteria?
16S rDNA is a strand of DNA from a bacteria

that is used and compared against a database
of all other bacteria strands of 16s rDNA
strands to find out which one the bacteria is
PART 1: SAMPLE PREPARATION

1. As the pathology lab technician, what is your task in this virtual lab?
Our task is to identify the bacteria strand using PCR and learn
the process of PCR through this process
2. Extracting DNA involves which initial step?

The initial step that extracts DNA is using a
digestive buffer to break down the cell wall
3. What is the wire ring used for?
The wire ring is used to transport the bacterial colony from the Petri
dish to the micro centrifuge tube
4. Why are the proteolytic enzymes necessary?
These enzymes are necessary because they

are the initial step that dissolves the cell wall
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5. Why do you then need to inactivate the proteolytic enzymes and how do you do it?
We need to use other enzymes later on in the process so we must inactivate the old enzymes
which are the proteolytic enzymes by heating the sample in a water bath that is 100c
6. After removing the enzymes, why do you spin the sample in the centrifuge?
You spin the cycle so that you can spin down the cellular debris
for removal from the sample
a. What is the pellet?
The cellular debris that is spun down the tube
b. What is the supernatant?
The liquid that the DNA is spun in that stays at the top
PART 2: PCR AMPLIFICATION

Go on to Part 2 and work through the PCR steps. Be sure to read the information in the notebook,
including “What is PCR?”
8. What does “PCR” stand for and what is the purpose of PCR?
PCR stands for Polymerase Chain Reaction and is a technique
that allows many copies of DNA to be made
Add the Master Mix and answer the following questions:

9. What does the Master Mix contain?
The mastery mix contains water, a buffer to keep ph the same,
large quantities of the four nucleotides and DNA primers that
bind the nucleotides to initiate the replication process
10. What are primers? Why is a primer added?
Primers are small prices of DNA that bind to specific sequences

in the bacteria 16s rDNA strand. They are added so when new
copies are made they overlap in the region of interest
11. Once the primers bind, what occurs next?
DNA polymerase extend the DNA from 5’ end to the 3’ end
12. What does "highly conserved" mean?

This means that all of the 16S rDNA strands are similar
amongst bacteria
13. Why are highly conserved regions important in this lab?
This is important because it allows them to use the same

primers even when they don’t know what bacteria they are
working with because is works with all of them
14. What does "highly variable" mean?
Highly veritable means that they are parts of the gene are
different
15. Why are highly variable regions important in this lab?
This is important because these regions are used for

identification between bacteria’s
Now run the PCR. Be sure to watch the virtual lab animation before proceeding to the questions.
16. After eight cycles, how many copies of the desired DNA have been synthesized?
After 8 cycles there are 240 copies of the DNA
PART 3: PCR PURIFICATION
17. Approximately how long is the 16s rDNA (bp)?
1500 base pairs(bp) long
PART 4: SEQUENCING PREPARATION

Click on "Learn about cycle sequencing before proceeding."
18. Read the first two paragraphs and list the steps in cycle sequencing in the space provided.
Step one is to use a thermocycle to create many copies of the

target DNA. You terminate the replication process at random
places so the copies are partial sequences with different
lengths.
Click to go back to Part 4.

19. What do the green and blue tubes contain? Describe the “sequencing brew” to which you added
your purified PCR.
These green and blue tubes contain sequencing grew which

contains buffer primers, DNA polymerase, nucleotides, and
fluorescence-tagged terminators
20. The purpose of the second PCR is not to create identical copies like the first PCR you ran. What is the
purpose of this PCR?
The purpose of the second PCR is not to create identical copies

but instead many copies of variable lengths
21. Where do scientists obtain primers to be used in PCR and in this technique?
The scientists obtain these primers to be used in PCR and in

this technique from a commercial source
Watch the virtual lab animation before proceeding to Part 5.
PART 5: DNA SEQUENCING

22. What is the final PCR product, the stuff contained in your 12 tubes?
The stuff in the 12 tubes is a mix oof DNA pieces of variable

lengths
Be sure to watch the virtual lab animation before proceeding to Part 6.
PART 6: DNA SEQUENCE ANALYSIS

Click on "Learn about the science behind sequence matching."
23. What is BLAST and how is it used?
Blast stands for Basic Local Alignment Search Tool and is used
to find which bacteria the DNA comes from
Click to go back to Part 6 and click on "Learn more about BLAST search results."
24. Explain what the "Score (bits)" means on an actual BLAST search result.
Score bits is the numerical value that is used to indicate if a

bacteria is a match or not
25. What does an E-value of 3 or less represent?
An E value of 3 or less then means that it is an acceptable

match
Click to go back to Part 6 and proceed through the instructions in the right-hand notebook window.
● Hints: "Ctrl A" will select all the data in the pop-up window, "Ctrl C" will copy it, and "Ctrl V" will
paste it into the NCBI website (large yellow box at the top of the BLAST search page).
● Follow the steps listed on the page and be patient. BLAST data can take a while to search.
● When the BLAST results appear, scroll down below the color key to the significant alignments,
and then go back to the virtual lab window (left) and follow the instructions.
26. What is the scientific name of the bacterium you sequenced?
Bartonella Henselae
27. Write a brief description of this bacterium in the space provided.
Various species of bartonella that are pathogenic to humans are

transmitted via a vector or directly from and animal resivoir

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INTRODUCTION

16S rDNA is a strand of DNA from a bacteria

PART 1: SAMPLE PREPARATION

2. Extracting DNA involves which initial step?

4. Why are the proteolytic enzymes necessary?

These enzymes are necessary because they

a. What is the pellet?

The cellular debris that is spun down the tube

b. What is the supernatant?

PART 2: PCR AMPLIFICATION

Add the Master Mix and answer the following questions:

10. What are primers? Why is a primer added?

Primers are small prices of DNA that bind to specific sequences

12. What does "highly conserved" mean?

13. Why are highly conserved regions important in this lab?

This is important because it allows them to use the same

14. What does "highly variable" mean?

15. Why are highly variable regions important in this lab?

This is important because these regions are used for

1500 base pairs(bp) long

PART 4: SEQUENCING PREPARATION

Step one is to use a thermocycle to create many copies of the

Click to go back to Part 4​.

These green and blue tubes contain sequencing grew which

The purpose of the second PCR is not to create identical copies

The scientists obtain these primers to be used in PCR and in

Watch the virtual lab animation before proceeding to Part 5.​

PART 5: DNA SEQUENCING

The stuff in the 12 tubes is a mix oof DNA pieces of variable

Be sure to watch the virtual lab animation before proceeding to Part 6.

PART 6: DNA SEQUENCE ANALYSIS

Score bits is the numerical value that is used to indicate if a

An E value of 3 or less then means that it is an acceptable

26. What is the scientific name of the bacterium you sequenced?

27. Write a brief description of this bacterium in the space provided.

Various species of bartonella that are pathogenic to humans are

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Click to go back to Part 4.

Watch the virtual lab animation before proceeding to Part 5.