Lab 5 Flashcards
What is the purpose of restriction enzymes?
To protect bacteria from invading foreign DNA by digesting the foreign DNA at a specific recognition nucleotide sequence (6bps).
What do restriction digests require?
- DNA
- Restriction enzymes
- Buffer
- Water
What is the purpose of the buffer in a restriction digest?
To maintain an optimal pH of the solution for the enzyme.
What is the purpose of the water in the restriction digest?
Provides volume and allows easier mixing of the DNA and buffer.
Why is the concentration of DNA in the sample good to know for restriction digests?
Allows us to know the amount of restriction enzymes required for complete digestion of the DNA.
What is one unit (U) of restriction enzyme activity?
The amount of restriction enzyme needed to completely digest (cut) one microgram (ug) of substrate DNA in one hour at the optimum temperature of the enzyme (around 37).
How is enzymatic activity of restriction enzymes measured?
Units of their activity; ability to cut DNA.
What does excess of enzyme result in?
Incomplete or non-specific digestion of DNA.
What is electrophoresis?
Charged molecules migrate in an electrical field.
What is the rate of migration determined by?
The size of the molecules and their electrical charge.
How does the migration rate of DNA molecules relate to their molecular weight?
Inversely proportional
What is the purpose of ethidium bromide or GelRed?
Intercalate with DNA to allow the bands to be observed under UV light.
Where does the DNA move to in gel electrophoresis?
Move to the anode (positive charged pole; red) since it has a negative charge.
Where does the loading dye move in gel electrophoresis?
Move to the anode (positive charged pole; red) since it has a negative charge.
Where does the GelRed or ethidium bromide move in gel electrophoresis?
Move to the cathode (negative charged pole; black).
Why is a DNA ladder added?
Estimates the sizes of your bands.
What restriction enzymes are used in lab 5 experiment?
Xbal (cuts at T-A) and NotI (cuts at G-C).
Where will the restriction enzymes cut the blue colony (without the insert)?
Cut at MCS
How many bands does the blue colony produce?
1
Where will the restriction enzymes cut the white colony (with the insert)?
Cut at MCS
How many bands does the white colony produce?
2 - one regular DNA and the other is the insert itself.
Why are the restriction enzymes added last?
Added to the sample at which it has an optimal reaction condition.
What does loading dye do?
Adds weight to your DNA samples, making them dense so they can sink to the bottom of the wells.
Why are there multiple bands for the uncut DNA?
Multiple cloning site so plasmid DNA might be supercoiled, nicked, or linear.
Why does the supercoiled DNA travel faster and farther down?
Since it’s tightly wrapped, it experiences less friction.
Why does the nicked DNA travel slower and smaller distances?
Open circular plasmids cause more friction.
