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MCB 301 Final Exam questions with 100% Correct & Verified Answers
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MCB 301 Final Exam questions with 100% Correct & Verified Answers
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MCB 301 Final Exam questions with 100%Correct & Verified Answers
What is AGS?
Arginine monohydrochloide, Glycerol, Sodium Sulfate (used for enrichement of ab-
producing bacterium)
Describe DNA extraction from a sample and it's key steps:
1) Centrifuge sample to form pellet consisting of all cell material (supernatent is
discarded)
2) Re-suspend pellet in lysis buffer (contains lysozyme which degrades thick PG cell wall
found in G+ bacterium. Triton X-100 is a detergent which disrupts the integrity of
membranes. EDTA chelates divalent metal ions which nucleases require, therefore
spontaneous DNA cleavage is prevented)
3) Incubate
4) Proteinase K is added which degrades protein released from the cells when fully lysed
in the next step
5) BDL buffer is added which is an alkaline solution which rapidly lyses the cells.
6) Ethanol is added to precipitate the nucleic acids in preparation for purifying by using
spin columns.
7) Solution is added to a spin column where the DNA binds to the wafer membrane;
solution is centrifuged and flow-through is discarded
8) HBC buffer is added to remove most of the protein from the DNA prep; spin and
discard flow-through
9) DNA Wash buffer is added to remove most of the RNA from the DNA prep; spin and
discard flow-through
10) Centrifuge again at high speed to remove residual buffer and prevent future
interference.
11) Elution buffer is added to unbind the DNA from the membrane found in the spin
column; spin and KEEP flow-through
12) Repeat step 11, then store at 4C.
Describe the PCR procedure highlighting the important reagents used.
- Where do the primers anneal to?
- Highly conserved region of the 16S rRNA gene (conserved region is at the ends of the
sequence)
, - Forward and reverse primers used to amplify sequence.
- PCR product is washed with spin column and DNA wash.
What is the purpose of doing the electrophoresis in the AB-producing bacteria
experiment?
1) To determine if the DNA was properly isolated
2) To determine if the PCR product is the proper size of the known sequence (~1.5kbp)
What do transposable elements (or transposons) typically include? (3 things)
1) Transposase gene
2) DNA sequences at both ends to interact with the transposase
3) Selectable marker (e.g. virulence factor or resistance to Ab)
T/F: Transposons only occur in prokaryotic organisms
False, it can also occur in eukaryotic organisms
What is a polar mutation? What other effects can occur from transposon insertion?
- Polar mutation causes the loss of expression of other genes within an operon that are
downstream the insertion site of the transposon
- Out-ward facing promoters on the transposons may activate the expression of genes
near the insertion site
What transposon is used in the "Transposon Mutagenesis" lab? What is it's length?
What is the name of the plasmid and what type of bacterium holds it (is the donor)
and why? What is the designation of the transposon?
- Mini-Tn5 (derived from original Tn5)
- ~1.5kbp
- pRL27, E. coli b/c it is a donor to a wide variety of recipient strains via conjugation
- Tn5-pRL27
Name all the important elements on the pRL27? How long is it?
- Transposon: oriR6K (site of autonomous plasmid replication, but requires Pi protein,
encoded by pir gene, which is not found on recipient cell, but found in the host) + aph
(encodes kanamycin resistance)
- oriT: site where DNA is nicked prior to conjugal transfer of the nicked DNA strand (all
transmissible plasmids require this site)
- tnp: encodes for transposase enzyme
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