Lab Quiz 2 Flashcards
Quiz
How many base pairs and genes does the single chromosome of the ecoli we worked with have?
4.5 x 10^6 base pairs
3000 genes
How many base pairs does the each vector (plasmid) that we manipulated have?
2000 to 8000
What is the goal of the lab?
To isolate a vector.
Why did we use rcf (relative centrifugal force instead of rpm?
Because rcf will be the same no matter which centrifuge we use - standardizes the experiment.
What was the buffer P2 used for?
The buffer contained SDS (sodium dodocyl sulfate) and NaOH
SDS lyses cells - turned clear blue
NaOH will denature chromosomes, protein and vector
What was the P1 buffer useful for?
The buffer had RNAse in it.
The buffer was added because we wanted to make sure when we completed the next steps that RNAse would be immediately available.
What did the N3 buffer provide?
It has salts in it.
Bring the pH down to 7.0 pH ish
Proteins will refold somewhat
chromosome will refold somewhat
vector will come together
Why do we centrifuge at the highest speed after the addition of the N3?
Because we want to separate the partially folded chromosome and protein and the rest of the solution (supernate) which still contains:
- vector
- base
- salts
- lipids
What is the purpose of putting the supernate in a separation column and spinning it for a minute?
We want impurities like the SDS and NaOH, salts and lipids and any other debris to go through while the vector will stick to the positive charge of the separation silica complex.
What is in the PE buffer and what is the purpose of adding it as well as spinning it for another min?
The PE buffer contains ethanol and the purpose of adding this is because we want to clean the vector that has stuck to the separation column in case some stuff is still stuck to it.
What is in the EB buffer and what is the purpose of adding it?
The EB buffer is negatively charged which competes with the thing that the vector is sticking to and after centrifuge, the vector goes to the bottom.
It is called the elution buffer for a reason.
What is a spectrophotometer?
It is an instrument that is used to analyze the amount of light that is absorbed or transmitted by a substance to tell us about the concentration of stuff in our solution.
Why did we use a Nanodrop Spec?
because we are working with tiny amounts substance.
We were hoping to get a value that was above 30 ng/uL
Why do we use the wavelength of 260 nm for the spectrophotometer?
DNA and RNA absorb UV light most effectively at a wavelength of 260 nm.
