Restriction Enzymes
(2X) Why do restriction enzymes need to be kept on ice?
Restriction enzymes are temperature-sensitive and lose activity when heated. Ice maintains the enzyme's stability and activity.
Which step (1st, 2nd, 3rd, or 4th) should the enzyme be added to the microcentrifuge tube?
The 4th step as it ensures enzymatic activity and function are preserved.
If lambda DNA is linear, how many times would the enzyme have to cut the DNA to generate 5 DNA fragments?
The enzyme would have to be cut 4 times.
Explain what Agarose is.
Gel matrix with tiny pores through which DNA migrates.
What do restriction enzymes do?
Cut DNA at specific locations (restriction sites/recognition sites).
Which step (1st, 2nd, 3rd, or 4th) should the water be added to the microcentrifuge tube?
The 1st step as it ensures the correct volume of water is added.
If lambda DNA is linear, how many times would the enzyme have to cut the DNA to generate 205 DNA fragments?
The enzyme would have to be cut 204 times.
How does gel electrophoresis work?
Separates DNA fragments by size using an electrical field.
What are enzymes named after? Give an example
Enzymes are named after the bacteria from which they are purified.
EcoRI is the first enzyme from Escherichia coli
HindIII is the third enzyme from Haemophilus influenzae
Which step (1st, 2nd, 3rd, or 4th) should the DNA be added to the microcentrifuge tube?
The 3rd step since the enzyme is designed to recognize and cleave the DNA.
(2X) If lambda DNA is circular, how many times would the enzyme have to cut the DNA to generate 1,083 DNA fragments?
The enzyme would have to be cut 1,083 times.
Would a shorter DNA fragment move faster or slower through the agarose gel than a longer fragment?
A shorter DNA fragment would move faster through the gel than a longer fragment.
Explain what blunt cuts are.
Enzyme cuts both DNA strands in the middle of the restriction site generating ends with no unpaired bases.
Which step (1st, 2nd, 3rd, or 4th) should the buffer be added to the microcentrifuge tube?
The 2nd step as it provides the solution with the appropriate pH and salt concentrations for enzymatic activity.
If lambda DNA is linear, how many times would the enzyme have to cut the DNA to generate 233,941 DNA fragments?
The enzyme would have to be cut 233,940 times.
(2X) Why do shorter fragments move faster than longer fragments?
Smaller fragments squeeze through the pores more easily, so they migrate the quickest.