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