Lab final Flashcards
what are the two thiings dtetergent due in DNA extraction?
- lyses the DNA (breaks down cell membrane)
- Denautures histones and seperates them in the DNA
what does isopropanol do to the extracted DNA?
makes DNA precipitate out of the extraction buffer
what does the 70% ethanol do to the extracted DNA?
washes off proteins, RNA, and lipids
what does the addition of NaOH do to affect the florescence of the DNA?
NaOH denautures the DNA so that double stranded DNA can;t interact with the dye.
How does the NaOh denauture the DNA?
NaOH removes protons from nitogenous bases of DNA because it is a strong base. This disrupts the hydrogen bonding necessary for double stranded stabilization
which plate do you expect to find bacteria most like the wildtype bacteria?
LB/-pGLO plate
why would the LB/-pGLO plate have the most wildtype bacteria?
DNA plasmid was not added to the -pGLO therefore expression of the gene encoding GFP is not present.
which plate would most likely have genetically transformed bacterial cells?
both LB/amp and LB/amp/ara plates
Why would the +pGLO plates have genetically transformed bacteria cells?
Because they both allow for bacterial growth
-LB/amp expresses an ampicilin resistance gene enabling growth from the resistance
-LB/amp/ara allows GFP gene transcription when arabinose is present
which plates should be compared to determine if any genetic transformation has occured?
LB/amp/-pGLO and LB/amp/+pGLO
why would you compare LB/amp/-pGLO and LB/amp/+pGLO to see if genetic transformation has occured?
the -pGLO plate wont express ampicilin gene and will not grow while the +pGLO plate will have resistance to ampicilin and will grow GFP
what is a control plate?
a plate that serves purpose as to compare the outcome of the experiment to a baseline and help interperet results
what is the control plate in this experiement?
LB/-pGLO
why is the LB/-pGLO the control plate?
because plasmid is needed for growth and this plate is not manipulated by amp or plasmid which means no change will occur
How is pGLO expression in the transformed bacteria being induced?
the araC is added to the ara plate which allows GFP gene transcription, followed by production of GFP= bright green color under blue light
what is the purpose of the CaCl2 transformation solution?
it promotes plasmid DNA to bind to surface of bacteria cells by neutralizing (-) charge
what is the purpose of the heat shock to the DNA?
it facilitates entry of DNA into bacterial cells, it makes membranes porous and changes fluitidy of lipids
what bacterial structures must the pGLO plasmids pass through in order to enter the bacterial cells?
the cell membrane and cell wall
what two things were done to help the plasmid enter the cell?
- added transformation solution with CaCl2
- heat shocked
why was the microcentrifuge tube incubated for 10 minutes before adding to agar plates?
to allow the cells time to produce beta-lactamase
would you expect to observe wildtype bacteria on plates that didn’t contain ampicillin or arabinose?
yes, LB/-pGLO would have wildtype bacteria because DNA of the plasmid was not expressed (nothing to make it change)
what information would the results on the LB/amp/-pGLO plate provide?
it showed no growth meaning the introduction of antibiotic ampicillin could not be broken down and there was no plasmid on the plate
what information would the results on the LB/amp/+pGLO plate provide?
bacteria can grow because plasmid was present and it was able to create a resistence to ampicilin and genes for GFP are expressed
would there be any major difference in the number of coloonies between the LB/amp/+pGLO plate and the LB/amp/ara/+pGLO plate?
no there would be no difference
