©Themoon EXAM SOLUTIONS
25/11/2024 09:27AM
BIOC 301 - Xmas Exam and Answers
What is the goal of PCR? - Answers✓✓Artificially replicate the process of natural DNA
replication to generate (amplify) many copies of the piece of DNA of interest
What components are needed to do a successful PCR? What do the components do? -
Answers✓✓- Add heat to break hydrogen bonds binding dsDNA, turns into ssDNA (also EtNa
lysis)
-Polymerase (Taq) reads DNA strand 5' to 3' for the assembly of complementary strands dNTPs
-Primers which allow polymerase to know where to start/stop reading (complement to strand)
-dNTPS which allow for complementary base pairs to be added (A,T,C,G) as read by
polymerase
What are steps of PCR thermocycler? - Answers✓✓-Denaturation, process which breaks H-
bonds of DNA template to form ssDNA
-Annealing, process which allows primers to bind to PCR template (ssDNA)
-Extension, process which adds base pairs as polymerase reads template strand
What happens at each PCR step? what temperature and how are they determined? What times
and how are they determined? - Answers✓✓- 30secs-1min of 95°C to denature template strand.
Doesn't take long for DNA to denature at high temperatures (everything else in PCR reaction
unharmed)
-30secs-1min annealing stage between 45-65°C depending on annealing temperature of
primers. Time enough to allow primers to bind but not long enough to cause non-specific
binding.
-1 minute at 72-74°C for every 1000bp of DNA we want to amplify for extension process. 72-
74°C is optimal temperature for polymerase activity.
, ©Themoon EXAM SOLUTIONS
25/11/2024 09:27AM
What are some things that can go wrong with PCR? - Answers✓✓Contamination, non-specific
binding, stop codon in primers, mastermix [] error, thermocycle program wrong
How do we know if PCR worked/failed? - Answers✓✓Run through agarose gel, if you see
bands at desired bp, PCR worked. If PCR failed, no bands, smears, not at desired location...etc
What is the purpose of DNA spectrophotometry? - Answers✓✓Checks the purity of the sample
(DNA)
What values are being measured during DNA spectrophotometry and what does each value
mean? - Answers✓✓- The absorbance at 260nm which represents the absorbance peak in the
UV spectrum for nucleic acids (DNA/RNA)
-The absorbance at 280nm which represents the absorbance peak in the UV spectrum for
aromatic amino acids.
How can the values obtained from DNA spectrophotometry be used to calculate the
characteristics of DNA sample? - Answers✓✓Take 260nm/280nm ratio to get DNA/protein
ratio in sample. Clean DNA has a ratio of 1.8-1.9. Higher ratios means RNA have contaminated
the sample and lower ratios means proteins have contaminated DNA sample. dsDNA at
concentration of 50ug/mL has absorbance at 1.0.
What way does DNA migrate in agarose gel electrophoresis? - Answers✓✓DNA is negatively
charged at neutral pH, therefore it moves from cathode (-) to anode (+)
What is the purpose of agarose gel electrophoresis? - Answers✓✓Standard method used to
seperate, identify and purify DNA, RNA, and proteins
25/11/2024 09:27AM
BIOC 301 - Xmas Exam and Answers
What is the goal of PCR? - Answers✓✓Artificially replicate the process of natural DNA
replication to generate (amplify) many copies of the piece of DNA of interest
What components are needed to do a successful PCR? What do the components do? -
Answers✓✓- Add heat to break hydrogen bonds binding dsDNA, turns into ssDNA (also EtNa
lysis)
-Polymerase (Taq) reads DNA strand 5' to 3' for the assembly of complementary strands dNTPs
-Primers which allow polymerase to know where to start/stop reading (complement to strand)
-dNTPS which allow for complementary base pairs to be added (A,T,C,G) as read by
polymerase
What are steps of PCR thermocycler? - Answers✓✓-Denaturation, process which breaks H-
bonds of DNA template to form ssDNA
-Annealing, process which allows primers to bind to PCR template (ssDNA)
-Extension, process which adds base pairs as polymerase reads template strand
What happens at each PCR step? what temperature and how are they determined? What times
and how are they determined? - Answers✓✓- 30secs-1min of 95°C to denature template strand.
Doesn't take long for DNA to denature at high temperatures (everything else in PCR reaction
unharmed)
-30secs-1min annealing stage between 45-65°C depending on annealing temperature of
primers. Time enough to allow primers to bind but not long enough to cause non-specific
binding.
-1 minute at 72-74°C for every 1000bp of DNA we want to amplify for extension process. 72-
74°C is optimal temperature for polymerase activity.
, ©Themoon EXAM SOLUTIONS
25/11/2024 09:27AM
What are some things that can go wrong with PCR? - Answers✓✓Contamination, non-specific
binding, stop codon in primers, mastermix [] error, thermocycle program wrong
How do we know if PCR worked/failed? - Answers✓✓Run through agarose gel, if you see
bands at desired bp, PCR worked. If PCR failed, no bands, smears, not at desired location...etc
What is the purpose of DNA spectrophotometry? - Answers✓✓Checks the purity of the sample
(DNA)
What values are being measured during DNA spectrophotometry and what does each value
mean? - Answers✓✓- The absorbance at 260nm which represents the absorbance peak in the
UV spectrum for nucleic acids (DNA/RNA)
-The absorbance at 280nm which represents the absorbance peak in the UV spectrum for
aromatic amino acids.
How can the values obtained from DNA spectrophotometry be used to calculate the
characteristics of DNA sample? - Answers✓✓Take 260nm/280nm ratio to get DNA/protein
ratio in sample. Clean DNA has a ratio of 1.8-1.9. Higher ratios means RNA have contaminated
the sample and lower ratios means proteins have contaminated DNA sample. dsDNA at
concentration of 50ug/mL has absorbance at 1.0.
What way does DNA migrate in agarose gel electrophoresis? - Answers✓✓DNA is negatively
charged at neutral pH, therefore it moves from cathode (-) to anode (+)
What is the purpose of agarose gel electrophoresis? - Answers✓✓Standard method used to
seperate, identify and purify DNA, RNA, and proteins
