Analyzing Gel Flashcards
What dye is added when the DNA is loaded into the wells? Why?
A loading dye is added to weigh down the sample to keep it in place within the well and to create a dye front
Why is the positive electrode at the bottom of the gel?
That is because DNA has an overall negative charge, thus going toward the positive electrode
What substance is added to stain the bands and how does it work?
Ethidium bromide is added to stain and fluoresce the bands. It works by intercalating into the bases of DNA.
What is the first step in analyzing a DNA agarose gel?
Measuring the migration distance of the bands for the molecular weight standards
What is the second step in analyzing a DNA agarose gel?
Plot the data on semilog10 paper to construct a standard curve
What is the third step in analyzing a DNA agarose gel?
Determine the sizes of the bands for the restriction enzyme digests that were electrophoresed
What is the fourth step in analyzing a DNA agarose gel?
Compare your results to what is expected/predicted based on the restriction enzyme map of the plasmid that was digested
What is a semilog paper?
Semilog paper has a normal scale on the horizontal axis and a log10 scale on the vertical axis
What does the vertical axis on semilog paper represent?
The size of the bands from the molecular weight markers in bp or kB
What does the horizontal axis on semilog paper represent?
The migration of the bands of the molecular weight markers in centimeters
What is each set of numbers on the vertical axis on semilog paper?
A 10-fold jump
What is the final result after plotting the molecular weight markers on semilog paper?
A standard curve to determine the sizes of the bands in the other lanes
How do you measure band migration properly?
