Original Document: Bacterial ID Lab at Howard Hughes Medical Institute
Teacher Notes: This site can take a while to do, not recommended for one class period, most can be done at home. Also recommend that you go over the site with students when giving the assignment to familiarize them. This is an ADVANCED LAB, most basic freshman lever biology classes do not need to cover PCR techiques.
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 to perform the following procedures:
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.
1. The first step in the extraction process uses a wire loop to do what? obtain a sample of bacteria
2. Why are the digestive enzymes necessary for DNA extraction? destroys cell wall so DNA can be obtained
3. The centrifuge separates the cellular content into two distinct layers. Describe
these layers and what is contained within them. supernatant contains DNA, the bottom layer contains cellular debris that is not needed
4. PCR stands for _______polymerase chain reaction____________
5. What is a primer? pieces of DNA that bind to specific regions, initiates replication
6.
Describe what happens during the 3 cycles
Melt - DNA chains are separated
Anneal - DNA is cooled so that the primers bind to their binding sites
Extend - DNA chain is extended by polymerase
7. After 30 cycles, how many copies of the initial DNA strand have been produced? 1 million
Why is this stage called "amplification"? because it amplifies (copies) a small amount of DNA
8. To confirm that the PCR worked, three lanes are run that contain 3 materials. What materials are in the 3 lanes?
Lane 1 ____water (control)__________ Lane 2 ____known sample_________ Lane 3 _______your sample________
9. What is the overall purpose of the purification stage? __remove contaminants from previous procedure, such as primers
10. Describe the “sequencing brew” that you added your purified PCR to. - contains buffers, primers, nucleotides and a florescent tag
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 PCR? to produce many sequences of varying lengths using primers to copy sections
12. What is the final PCR product, the stuff contained in your 12 tubes? PCR product, a mix of DNA of variable lengths
13. What is gel electrophoresis? separates molecules of differing size, using gel and electricity
14. What is BLAST? Basic Local Alignment Search Tool, used to ID organisms from DNA sequences
15. What is the identity of the bacteria in your sample? Follow the steps listed on the page and be patient. BLAST data can take a while to search. Bartonella henselae
Extension and Formative Assessment
Rubric (checklist)
| 1 | 2 | 3 | |
| Usefulness of PCR is discussed (applications toward determining cause of illness or for identifying DNA) | |||
| Techniques are discussed, such as separating DNA, amplifying DNA, using primers | |||
| Discussion of automatic DNA sequencer, electrophoresis and how sequences of bases are obtained and identified | |||
| Overall prose, consistency, grammar, support, etc | |||
| Total Point (possible 10) | |||