Molecular Cloning

Question 1: As we discussed before, the RAD52 gene can be isolated from the yeast cells. What
should we do first so we can isolate the gene we need?
✓ Perform DNA extraction
– Use a restriction enzyme
– Separate the DNA using gel electrophoresis
– Perform DNA sequencing

Question 2: In order to isolate RAD52, what should we do after extracting all DNA from the yeast
cells?
✓ Amplify the RAD52 gene
– Transform the yeast cells
– Sequence the DNA
– Proceed to the ligation step

Question 3: There is a plasmid containing eGFP on your workbench.

How can we cut the eGFP gene from the plasmid?

✓ Perform restriction enzyme digestion
– Insert the plasmid into bacteria so they secrete the gene
– Isolate the DNA using phenol-chloroform extraction
– Perform DNA sequencing on the whole plasmid

Question 4: How can you confirm that we have successfully extracted the DNA from the yeast?
✓ Analyze it using gel electrophoresis and the NanoDrop
– Analyze it using the UV transilluminator
– Weigh it using an analytical scale
– Analyze it using gel electrophoresis and an analytical scale

Question 5: After performing the PCR with primers that are specific for the RAD52 gene, we can
perform gel electrophoresis and compare the RAD52 DNA with a ladder.
Click on the 'View Image' button to see the gel electrophoresis result.

What can you conclude about the size of the RAD52 gene?
✓ The RAD52 gene is about 1500 bp long
– The RAD52 gene is precisely 1416 bp long
– The RAD52 gene is precisely 2543 bp long
– The RAD52 gene is about 3500 bp long

Question 6: Where is the eGFP gene located?

You can find this information by clicking the 'View Image' button and view the plasmid map.
✓ At position 2506-3225
– At position 1-267
– At position 4670-4890
– At position 702

Question 7: Which two restriction enzymes cut this plasmid once and can be used to isolate the
eGFP gene?

Click the 'View Image' button and find the single restriction sites.
✓ XbaI and XhoI

Page 1
 – NcoI and XhoI
– NotI and EcoRI
– We only need to use one enzyme

Question 8: In order to ensure that the restriction enzymes work properly, we need to select the
optimal buffer and incubation temperature!

Which buffer should we choose for cutting the eGFP using XbaIXba 1 and XhoIXho 1?

Click the 'View Image' button to see a table containing the ideal conditions for each restriction
enzyme.
✓ Buffer 3
– Buffer 4
– Buffer 2
– Buffer 1

Question 9: Which incubation temperature is optimal for cutting the eGFP from its plasmid?
Remember we are using XbaIXba 1 and XhoI to isolate the eGFP. Click the 'View Image' button to
see the table.
✓ 37 °C
– 80 °C
– 65 °C
– 25 °C

Question 10: You have just learned about the three types of ends created on DNA fragments by
restriction enzymes. Which of the following restriction enzymes produces blunt-end DNA strands?
Click on the 'View Image' button to see the resulting DNA sequence for each of the restriction
enzymes.
✓ EcoRV
– XhoI
– EcoRI
– HindIII

Question 11: What is the next step after performing gel electrophoresis to obtain the pure eGFP
fragment that has been cut from its plasmid?
✓ Perform an electrophoretic gel extraction
– Perform restriction enzyme digestion
– Perform enzymatic DNA Ligation
– Perform spectrophotometric NanoDrop analysis

Question 12: Click 'View Image' to see the gel electrophoresis result. The DNA ladder is in well 1.
In wells 2-7, you and the assistant have loaded a variety of digested plasmids.

eGFP was isolated from the plasmid pPyCAG-eGFP-IP using XbaIXba 1 and XhoIXhol 1 restriction
enzymes. Based on this information, which band contains the eGFP?
Hint: eGFP is 720 bp, pPyCAG-eGFP-IP is 6660 bp.
Hint: eGFP (720 bp), pPyCAG-eGFP-IP (6660 bp).
✓C
–D
–B
–A

Question 13: There are several possible outcomes when performing ligation. Think about which
restriction enzymes were used to isolate the two genes. Click on the 'View Image' button for
reference.

Page 2
Which of these DNA orientations is correct when ligating RAD52 and eGFP?

✓ Figure A
– Figure D
– Figure C
– Figure B

Question 14: Which of the following techniques are we going to use in order to insert exogenous
DNA into the yeast cells?
✓ Transformation
– All of the options
– Transduction
– Conjugation

Question 15: We know that the exogenous DNA cannot enter the yeast cell by itself. What should
we do to make the transformation successful?
✓ Create competent yeast cells
– Add antibiotic to the medium
– Make the growth medium richer
– Amplify the exogenous DNA

Question 16: It is common that only some of the yeast cells will successfully take up the plasmid.
How do we determine which yeast colonies contain the pTRE-RAD52-eGFP plasmid?
✓ Through antibiotic selection
– Through gel electrophoresis
– It cannot be determined
– By sequencing each colony

Question 17: Which colony should we pick for the expression analysis?

You can access the transformation results by clicking 'VIEW IMAGE' button.

Page 3
 ✓A
–D
–C
–B

Question 18: Why should we pick a single colony?

✓ All answers are correct
– To avoid large genetic variance in the expanded culture
– To ensure all cultured yeast cells are identical
– To ensure all cells grown are genetic clones of a single original cell

Question 19: Which of the following images would you expect when you have inoculated and
incubated the transformed yeast in a broth medium? Click the 'View Image' button to see the
images.
✓ Picture I because the yeast cells multiplied in the medium
– Picture I because the yeast cells did not multiply in the medium
– Picture II because the yeast cells multiplied in the medium
– Picture II because the yeast cells did not multiply in the medium

Question 20: In which phase should we start to expand the yeast culture into a new agar plate?
✓ When the yeast culture reaches the log phase
– When the yeast culture reaches the late-lag phase
– When the yeast culture reaches the stationary phase
– When the yeast culture reaches the lag phase

Question 21: How can the RAD52-eGFP gene constructkahnstruct help us to study the DNA repair
mechanism?
✓ We can observe the eGFP
– We know the DNA sequence of eGFP
– Transformed yeast will grow better in UV light
– Analyze the RAD52 gene with sequencing

Page 4