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Natural Science

4. How many fragments would produced if pGLO was digested with HindIII and BamHI

4. How many fragments would
produced if pGLO was digested with HindIII and BamHI? Use a diagram of the
digests in your answer. 2 marks
There would be 5 fragments.
HindIII cuts twice and BAMHI cuts thrice, leaving 5
fragments.
5. Say you wanted to use pUC18 (instead of pGLO)
for Practical 4. Would you be able to use the BseYI (instead of HindIII)
restriction enzyme for the digest?
Why/why not? 1 mark
TBA
6. Say you wanted to use pUC18 (instead of pGLO)
for Practical 4. Can you still use arabinose to induce the cells to express BSA
(like in Practical 6)?
Why/why not? 1 mark
7. Would the insert still be present in the
recombinant plasmid for Practical 4 if the transformed cells were plated on
agar plates (Step 20 – 24) containing ampicillin only (no arabinose)? Why/why
not? 1 mark
8. 2 part question: Say the PCR result for
Practical 4&5 confirmed the presence of an insert in the recombinant
plasmid (pGLO vector), but there was no BSA protein expressed after arabinose
induction.
a. What would be the source
of this problem? 1 mark
b. What would you change in the methods to
ensure it doesn’t happen again?
1 mark
9. 2 part question:
a. Would you still amplify the target ALB gene
in Practical 5 if the Extension time
(Step 3) was changed to 5 min (instead of 2.5
min)? Why/why not? 1 mark
b. If you ran the PCR products on the gel, what
approximate sizes would the
bands be? 1 mark
10. 2 part question:
a. What would happen to the proteins if SDS
detergent was not used in the “Lysis
Buffer” and “Sample Buffer” for Practical 6? 1
mark
b. Would excluding the SDS detergent affect the
SDS-polyacrylamide gel
electrophoresis? Why/why not? 1 mark