laboratory: unit 3: gram stain microscopy; inoculate broth (pages 52-53) lecture: pcr ribosomal...

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

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

Identification of bacterial speciesby PCR amplification

and sequencing16S ribosomal RNA genes.

Zone of clearingaround colonyindicates antibioticproduction.

Antibiotic-producing colony of Bacillus subtilis

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.

double-stranded template DNA

94oC

50oC

denature

anneal primers

72oC DNA polymerase

+

+

+

Cycle 1

94oC

50oC

denature

anneal primers

72oC DNA polymerase+

+

Cycle 2 +

+

94oC

50oC

denature

anneal primers

72oC DNA polymerase+

Cycle 3

+

+

+

94oC50oC

denatureanneal primers

72oC DNA polymerase

Cycle 4

Cycle 5

16S ribosomal RNA(partial sequence)

PCR primers:27F & 519R

Purify PCR product prior to sequencing

binding buffer (PB) = high salt

wash buffer (PE) = high salt + ethanol

Purify PCR product prior to sequencing

50 ul distilled water

dissolve in 50 ul distilled water

ready for sequencing