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Notes taken during lab


Department
Molecular Genetics and Microbiology
Course Code
MGY299Y1
Professor
Johanna Rommens

Page:
of 2
Lab 2 September 22, 2011
Phage: bacteria-infecting virus
Titre: concentration of phage
- units: NPF/mL
Amber mutation: nonsense mutation => premature stop codon in essential gene
- no protein for phage to replicate
- phage with amber mutation => no plaques, all bacteria
Amber suppression: suppress amber mutation so phage can grow
- tRNA recognizes amber codon UAG and insert amino acid instead of terminating
protein
- what this means for the rest of the cell
obecause many genes won’t terminate, not all stop codons are amber
codons
redundant at end, to stop properly
Repressor: DNA binding protein regulating/blocking transcription
Reverent: bacteria acquiring mutation to revert back to wild-type phenotype
Lots of bacteria plated => cloudy agar plate
Phage lytically in bacteria => plaque/clearing on plate
- Each plaque considered to have come from one phage: genetically pure
How could phage with amber mutation grow?
- amber suppressor
- reverents
Serial dilution
10-6 = 1/(106) = 1 000 000 fold dilution
10-1 = tenfold dilution
10-1 10-3 10-4 10-5 10-6
4.5mL 9.9mL 4.5mL 4.5mL 4.5mL
Exercise from lab 1:
0.5mL
phage
0.1mL 0.5mL 0.5mL 0.5mL
Minimal plates – are genes required for vitamin synthesis intact?
MacConkey plates – are genes required for sugar metabolism intact?
EX. leu and gal strain
- minimal medium => no growth
- minimal + leucine => growth
- MacConkey gal => white
- MacConkey lac => red
oCan metabolize lactose
Lots of bacteria plated => cloudy agar plate
Phage infecting bacteria lytically => plaque/clearing on plate
- each plaque considered genetically pure b/c originated from 1 phage