Methods Flashcards
(21 cards)
what is bottleneck?
it refers to events that sharply limit population size such as transferring to another culture usually in experimental evolution. we had a 1:100 bottleneck.
Why do we put MH4 in ampicillin and then transfer to LB? And then doing the same but pass in LB + bleomycin?
when we pass it LB we expect it to be none remaining after a few generations. And doing the same in Amp we expect 100% remaining. But when doing it in bleomycin we hope it can stabilize it so that 50-100% is remaining. usually we dont care about bleMBL, but if we can prove that this can stabilize it and select for it has a high clinical relevance
Why is there troublesome to have few replicates in relative growth rate?
It corresponds to much noise and error bars and therefore we increased number of replicates
What is the red area in the curve in BAT?
it relates to enough variation to make big error bars
In relative growth rate if we get 0,86, what does that mean?
it means it has a 14% fitness cost so its more costly
In BAT what is the R value?
We want to see linear relation and therefore we change the R-value to have linear data
R-value is about the linear correlation between time and OD for the fitted line. if the system cant find the log phase we have to manually adjust the settings so in theory the value should be 1 but its always lower because of measurement errors.
what is the lag phase in the relative growth rate?
this is the phase where growth is abscent which is followed by an acceleration phase until entering a constant growth rate is achieved during the exponential phase. eventually it decline during the deceleration phase and growth ceases due to resource echaustin and waste accumulation during the saturation phase.
why did we choose pbad18?
because of availability
what enzymes did we start with and why did we change them? We ended up with Xbal and SalI
We started with Xbal and SmaI
What is important to remember when using blunt ends?
You have to phosphorylate the inserts to make phosphodiester bonds between the insert and the vector
Why do we do a digestion?
To remove extra basepairs
Why do we have a water control in the ligation?
To see how many plasmids there are , essentially the background
Why do we want the overnight cultures on shaking?
To ensure oxygen and enough room for growth and therefore the cork on the culture tubes are loose to ensure oxygen-rich conditions
why did you mix 2 ul sample and 10 ul loading dye dilution and not the standard 10 ul sample + 2 ul of 6x loading dye
because its standard in our lab to do it this way to save PCR products for downstram use
why did we start with blunt ends first and why did we switch?
we wanted to start with sticky ends but due to the restriction enzyme sites in the vector and the inserts we had to use blunt end. when the enzyme chosen were not working we had to change tacticts and can then swithch over to sticky end
why do we want to use sticky end ligation over blunt end ligation?
its higher efficiency, the parts stick together before the ligase seals them so its easier and more specific and because of overhangs one can control the direction
what were we looking for in the sanger sequencing?
to always get the correct orientation of the inserts, however with sticky end we were only looking for point mutations, but in blunt ends we would also be looking for correct orientations
Why did we use low salt LB?
if there is too much salt in the solution the machine will arc and thats why we use low salt LB and wash them twice to prevent arc
What is FACS
it stands for fluorescence activated cell sorting and makes it so i can analyze and physically separate specific cells based on their fluorescent properties
What does the clinical breakpoints for eucast mean?
The clinical breakpoint is defined by a critical MIC and is used to determine whether a bacterial strain is susc or res to a particular antibiotic in a clinical setting
