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BIOL 342 Midterm & Final Exam Questions with Correct Answers
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You want to clone a DNA fragment that has been digested with BamHI into a plasmid vector. What should you do to prevent religation of the vector? If you don't do this what other problems will occur? Correct Answer-To prevent religation you should treat it with alkaline phosphatase which will knock ...

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BIOL 342 Midterm & Final Exam Questions with
Correct Answers
You want to clone a DNA fragment that has been digested with BamHI
into a plasmid vector. What should you do to prevent religation of the
vector? If you don't do this what other problems will occur? Correct
Answer-To prevent religation you should treat it with alkaline
phosphatase which will knock off the 5' phosphate. As a result you will
have 5' and 3' OH which means ligase can't act on it.


If you don't, you could get a concatemer or the insert could go in upside
down or backwards.


Describe five characteristics of a plasmid cloning vector Correct
Answer-1) Origin of DNA replication
2) Selectable marker
3) Unique restriction endonuclease sites
4) Small size
5) High copy number


T/F


E.coli cells transformed with the cloning vector pUC19 (w/o insers)
produce white colonies in the presence of X-gal and when grown in
IPTG and ampicillin Correct Answer-F

,They produce blue colonies because they are functional as they do not
contain an insert


Why is cloning using a double-digestion preferred over using a single
REN site? Give three reasons Correct Answer-1) Prevents self-ligation
(two different RE cut sites won't match up like one will)
2) Allows for directional cloning (two different REN sites means only
one possible direction)
3) Prevents insert-insert and vector-vector concatemers


What are the advantages of using pUC19 over pBR322? Correct
Answer-- MCS -> wide choice of REN sites for cloning and the sites are
adjacent to one another so you have more options of using 2 different
REN sites, this in turn facilitates the prevention of self-ligation and
directional cloning as the insert can only go in one way
- Small (2.6kb vs 4.3kb pBR322) -> can facilitate more efficient
transformation and this is allows for high copy number
- lacZ' blue-white differentiation -> no replica plate needed therefore it
saves time


Explain the steps in the phosphoramidite method of automated chemical
synthesis of DNA Correct Answer-1) Release of 5' DMT group from 1st
nucleoside by acid hydrolysis, this leaves the 5' OH group ready for
addition
2) Coupling step where covalent bond between 3' phosphite of incoming
phosphoramidite and 5' OH of attached nucleoside is formed

, 3) Unreacted 5' OH groups must be blocked, these are all capped by
acetylation
4) Resulting phosphite triester bond is oxidized to form a more stable
phosphate triester bond
5) Methyl groups are removed and chains is cleaved from the spacer
molecule leaving a 3' OH end
6) DMT is removed and 5' OH end is phosphorylated
7) Full length chains are purified from truncated chains by
chromatography


What are disadvantages of Sanger/Dideoxy sequencing? Correct
Answer-- Not real time -> needs extra gel electrophoresis step


T/F


The forward primer has the same sequences as the top strand and binds
to the bottom strand Correct Answer-T


What is the difference between a primer and a probe? Correct Answer-
Primer - used to initiate DNA synthesis


Probe - modified pieces of DNA that are able to detect specific
sequences in a mixture


T/F

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