Mutagenesis - Experiments 4 and 5 Flashcards
What is the difference between a spontaneous mutation and an induced mutation?
Spontaneous mutations are completely random and happen without any exposure to environmental agents. Induced mutations are cause by an environmental agent
What is a mutation?
A change in the DNA that affects a gene and its function
What is a forward mutation?
Wild-type -> mutant. Results in a mutant phenotype
What is a reverse mutation?
Mutant -> wild-type. Results in the restoration of the wild-type phenotype
Why are reverse mutations more rare than forward mutations?
Reverse mutations require a precise change in the genetic code to get it to go back to wild-type
Why do we spread millions of auxotrophic cells to measure the mutagenic effect of UV light?
Because reverse mutations are so rare, it takes millions of cells to have decent odds of getting a revertant
Why isn’t it sufficient to only count the number of revertants to determine the rate of reversion?
Mutagens also have a killing effect, and the killing effect needs to be factored in
What medium is used to measure the mutagenic effect of UV?
Minimal media. Any revertants will grow, any that don’t revert won’t grow
What medium is used to measure the killing effect of UV?
Complete media. All cells should grow on complete media, so it is easy to measure how many cells are dying
Why do we only spread a few hundred cells on the complete media plates to measure the killing effect? (3 reasons)
All cells should grow on complete media, so we need to have a small enough number that we can still count. We also need to minimize statistical errors with the small sample size and avoid overcrowding the colonies
Why do we use serial dilutions?
Eliminates the need for very large amounts of the diluent so we get a much smaller and more manageable volume
What parameters are used to calculate Cv in Cv=S0/(Ds x V)? Why is this being calculated?
Cv: cell concentration of the original culture
S0: number of cells on the unirradiated plate
Ds: the dilution used for the supplemented plates
V: the volume plated (usually 0.1 ml)
We need to know how many cells were in the culture before dilution
What parameters are used to calculate Ns in Ns = Cv x Ds x V? Why is this being calculated?
Ns: Number of viable cells plated on the YEPD plates
Cv: cell concentration in the original culture
Ds: dilution used on the supplemented plates
V: volume plated
This tells us how many cells were spread on each YEPD plate
What parameters are used to calculate Nm in Nm = Cv x Dm x V? Why is this being calculated?
Nm: number of viable cells spread on the YM plates
Cv: cell concentration of the original culture
Dm: the dilution used on the YM plates
V: the volume plated
This tells us how many cells were spread on each YM plate
What parameters are used to calculate Pt in Pt = St/Ns? Why is this being calculated?
Pt: the proportion of survivors at time t
St: the number of cells on the supplemented plate irradiated for t seconds
Ns: the number of viable cells spread on the YEPD plates
This tells us the killing effect, tells us what the proportion that should survive the dose of UV light at that amount of time
