Question

                                               

Bacterial Identification by Molecular Methods

Type your responses in blue.

Go to the Howard Hughes Medical Institute educational website and open the “Bacterial Identification Lab” (http://www.hhmi.org/biointeractive/bacterial-identification-virtual-lab). Click on "Launch Interactive." Perform the entire simulated lab exercise. Be sure to read all of the instructions and background information provided along the way. Type your answers in blue and include with your Cloning Lab Write-Up.

1. Which specific gene is used to identify the unknown bacteria?

2. How is the bacterial DNA extracted from the cells? How is the DNA separated from the cellular debris?

3. What is PCR? What is the source of the special DNA polymerase that is used in PCR (name the organism (genus and species) and its natural habitat)?

4. What is contained in the PCR Master Mix? What should happen at each of the 3 temperatures cycled: 95^o C, 60^o C and 72^o C?

5. About how many total copies of the bacterial gene should there be at the end of the 30 cycles? About how long (in base pairs) should the PCR product be?

6. Why are the fluorescent dideoxynucleotides called terminators? Why were 12 primers used in the sequencing step?

7. What is the purpose of the gel electrophoresis performed in the sequencer machine? What does BLAST stand for?

8. Use the NCBI site to compare the gene sequences and identify the genus and species of each sample. Write the organisms' names below (should be 6 different species):

Sample A (lymph aspirate)

Sample B (stool sample)

Sample C (urine sample)

Sample D (blood sample)

Sample E (sputum sample)

Sample F (stool sample)

Answer 1