1. Laboratory: Unit 3: Gram stain & microscopy; inoculate broth (pages 52-53) Lecture: PCR & ribosomal RNA-based phylogeny In-Class Writing: describe colonies (page 51); peer review lab report 2 (209-210) Hand In: lab report 2 draft (pages 37-39) Read: manual (pages 52-53); Day, chapter 37 Due Next Class: lab report 2; flow chart 3

2. Due Class 9: lab report 2 (pages 207-208) Send your lab report to:

4. Identification of bacterial species by PCR amplification and sequencing 16S ribosomal RNA genes.

5. Zone of clearing around colony indicates antibiotic production.

6. Antibiotic- producing colony of Bacillus subtilis

7. Polymerase Chain Reaction (PCR) Amplify specific DNA from complex mixture. PCR is: repetitive primer-directed DNA synthesis using heat-stable DNA polymerase to extend primers annealed to opposite strands of template.

8. double-stranded template DNA 94 o C 50 o C denature anneal primers 72 o CDNA polymerase + + + Cycle 1

9. 94 o C 50 o C denature anneal primers 72 o CDNA polymerase + + Cycle 2 + +

10. 94 o C 50 o C denature anneal primers 72 o CDNA polymerase + Cycle 3 + + +

11. 94 o C 50 o C denature anneal primers 72 o CDNA polymerase Cycle 4

12. Cycle 5

13. 16S ribosomal RNA (partial sequence) PCR primers: 27F & 519R

14. Purify PCR product prior to sequencing binding buffer (PB) = high salt wash buffer (PE) = high salt + ethanol

15. Purify PCR product prior to sequencing 50 ul distilled water dissolve in 50 ul distilled water ready for sequencing