Bacterial Identification by MolecularMethods

Type your responses in blue.

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

1. Which specific gene is used to identify the unknownbacteria?

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

3. What is PCR? What is the source of the special DNA polymerasethat is used in PCR (name the organism (genus and species) and itsnatural habitat)?

4. What is contained in the PCR Master Mix? What should happenat 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 shouldthere be at the end of the 30 cycles? About how long (in basepairs) should the PCR product be?

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

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

8. Use the NCBI site to compare the gene sequences and identifythe genus and species of each sample. Write the organisms' namesbelow (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)