1) Suppose we used the following two restriction enzymes for cloning: (1) BamH1
ID: 173756 • Letter: 1
Question
1) Suppose we used the following two restriction enzymes for cloning:
(1) BamH1 which recognizes the sequence (2) Sau3A which recognizes
the sequence
GGATCC GATC
CCTAGG CTAG
to produce 5'-sticky ends with a 4 single- to produce 5'-sticky ends
stranded base overhang. with a 4 single-
stranded base overhang.
Diagram/draw the sticky ends produced.
(c) (4 points) The vector DNA to be used for cloning is digested with BamH1 and the target DNA used for cloning is digested with Sau3A. Would using these two enzymes as described work for a cloning experiment? Explain.
(d) (2 points) - What is the name of the enzyme used to join the ends of the target and vector DNA molecules together?
(e) (4 points) - Why was it necessary to treat the vector with phosphatase?
(f) (6 points) - What would happen during the ligation reaction if the vector sample was not heat inactivated before the ligation reaction?
(g) (12 points) - Explain how the blue/white colony selection works. (In other words, after the transformation, you obtain blue and white colonies on the agar plate. How/why were you able to create these blue/white colonies.)
(h) (4 points) - If the phosphatase didn't work, explain how this would affect the relative number of blue and white colonies.
(i) (6 points) - If the restriction enzyme only digested half of the circular vector molecules to linear molecules, explain how would this affect the relative
number of blue and white colonies.
Explain how you could get pale blue colonies?
Explanation / Answer
(c) (4 points) The vector DNA to be used for cloning is digested with BamH1 and the target DNA used for cloning is digested with Sau3A. Would using these two enzymes as described work for a cloning experiment? Explain.
In cloning experiment, it is normal to use two restriction endonuclease to cleave the vector as well as the gene of interest to get the desired product an
(d) (2 points) - What is the name of the enzyme used to join the ends of the target and vector DNA molecules together?
The enzyme used to join the end of the target and vector DNA is DNA ligase
(e) (4 points) - Why was it necessary to treat the vector with phosphatase?
Since the vector is digested by BamH1 due to their is the possibility of getting it self-ligated. Treatment with phosphatase will remove phosphate due to which vector will not be able to self ligate
(f) (6 points) - What would happen during the ligation reaction if the vector sample was not heat inactivated before the ligation reaction?
BamH1 enzyme will show some activity on the vector molecule and continue to digest it. in order to deactivate the enzyme we need to heat activate the enzyme
(g) (12 points) - Explain how the blue/white colony selection works. (In other words, after the transformation, you obtain blue and white colonies on the agar plate. How/why were you able to create these blue/white colonies.)
(h) (4 points) - If the phosphatase didn't work, explain how this would affect the relative number of blue and white colonies.
(i) (6 points) - If the restriction enzyme only digested half of the circular vector molecules to linear molecules, explain how would this affect the relative
number of blue and white colonies.
Explain how you could get pale blue colonies?
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