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BCH5413 Exam One 2024 with Questions and 100% Correct Answers
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BCH5413 Exam One 2024 withQuestions and 100% Correct Answers
in a cloning experiment, what does it mean when you get colonies on a No ligase / No
insert control plate? - Answer This means that your plasmid was either incompletely cut
by the endonuclease or it somehow recircularized itself.
What if there are more colonies there than on your experimental? - Answer Thats not
good if there are more there than on your experimental because likely you don't
have any samples with you plasmid in it.
In what two ways did the development of the TOPO cloning vector make cloning easier?
- Answer One step combination of the plasmid and insert (taq pcr product) and
topoisomerase will link the DNA insert into a region with the multiple cloning site.
What is a shuttle vector? What additional features (beyond #2) would you want in a
shuttle vector? Why? - Answer Do your cloning in e.coli then shuttle it into a different
host cell system. Also want yeast origin and centromere so that the other host system
can recognize the plasmid and replicate it. Also want a selective marker for the yeast.
In creating a cDNA library what is the advantage of using beads containing oligo T
nuceotides for isolating the RNA? - Answer Used to isolate mRNAs because they
will anneal to the poly a tail
,In creating a genomic library, what is the advantage of using a partial digest over a full
digest for creating fragments for cloning? - Answer You get overlapping fragments that
increase chance to be able to identify a clone. Also good if you need to alter time.
Explain library screening using plaque hybridization. - Answer Take plate with plaques
and touch it with a filter. Block filter with radioactive protein and detect by
autoradiography.
When attempting to clone a specific cDNA, what are degenerate primers, AND
why might they
be advantageous? - Answer Comes in handy if you are not certain about which sequence
you want to amplify via PCR. Basically a way to have all different amino acid
combinations in the primer so that one will annex to sequence of interest.
What is a reporter gene? AND, why might it be useful in studying the characteristics of a
newly cloned gene? - Answer A reporter gene is used to test DNA regulatory elements.
Is useful because it will show you have the new cloned gene is acting.
With reference to ChiP: how are cells cross-linked - Answer cells are treated
with formaldehyde
With reference to ChiP: how is the chromatin sheared - Answer mechanically
With reference to ChiP: which antibody is used as a control - Answer IgG
With reference to ChiP: which two ways in which the chromatin obtained from ChiP to
be quantified? - Answer PCR using gene specific oligonucleotide primers, agarose gel
electrophoresis
, In Maxam & Gilbert Sequencing: Why do you need to radioactively label that fragment
that is being sequenced? - Answer So that you can be certain it is your fragment
In Maxam & Gilbert Sequencing: Why do you need to chemically modify the bases? -
Answer So that they could be cleaved at specific locations
In Maxam & Gilbert Sequencing: Why do you need an acrylamide gel? - Answer So
you can separate the fragments out by size to single nucleotide differences
In Sanger Sequencing: Why do you need ddNTPs ? - Answer They terminate the chain
In Sanger Sequencing: Why do you need a primer? - Answer To create a
complementary strand of DNA that you will sequence
In Sanger Sequencing: How does the automated approach reduce the number of
reactions from 4 to 1? - Answer All four reactions could be run together in one
lane because the fluorescent markers would all be a different color
In Sanger Sequencing: How are the different bases detected in capillary electrophoresis
as compared to normal gel electrophoresis? - Answer You see the different colors in
the peaks in the gel`
In next generation sequencing, what is the advantage of adapter sequences ligated to
DNA fragments - Answer No longer needed to run gels, you can use the adaptor to
start primer extension
Compare and contrast the method of base detection between pyrosequencing and ion
torrent sequencing. - Answer Pyrosequencing you add first base to well and if it is not
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