What is the goal of PCR? - ANSWER 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? - ANSWER - Add heat to break hydrogen bonds binding dsDNA, turns
into ssDNA (also EtNa lysis)
-Polymerase (Taq) reads DNA strands 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 allows for complementary base pairs to be added (A,T,C,G) as
read by polymerase
What are the steps of a PCR thermocycler? - ANSWER -Denaturation, process
which breaks H-bonds of DNA templates 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? - ANSWER - 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.
What are some things that can go wrong with PCR? - ANSWER
Contamination, non-specific binding, stop codon in primers, mastermix [] error,
thermocycle program wrong
, How do we know if PCR worked/failed? - ANSWER 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? - ANSWER Checks the
purity of the sample (DNA)
What values are being measured during DNA spectrophotometry and what does
each value mean? - ANSWER - 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? - ANSWER 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? - ANSWER
DNA is negatively charged at neutral pH, therefore it moves from cathode (-) to
anode (+)
What is the purpose of agarose gel electrophoresis? - ANSWER Standard
method used to seperate, identify and purify DNA, RNA, and proteins
What components are needed for successful gel? What does each component
do? - ANSWER -TBE buffer in which the Tris component is a basic salt and
provides conductivity. Boric acid lowers pH of buffer, so DNA does not
hydrolyze, maintain correct charge to migrate through gel.
-EDTA is a chelating agent, binds to divalent cations like Mg2+ which is often
required by enzymes to degrade DNA
-Loading buffer to visualize the bands (blue/purple).
-DNA ladder to determine size (bp) of fragments in gel.
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? - ANSWER - Add heat to break hydrogen bonds binding dsDNA, turns
into ssDNA (also EtNa lysis)
-Polymerase (Taq) reads DNA strands 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 allows for complementary base pairs to be added (A,T,C,G) as
read by polymerase
What are the steps of a PCR thermocycler? - ANSWER -Denaturation, process
which breaks H-bonds of DNA templates 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? - ANSWER - 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.
What are some things that can go wrong with PCR? - ANSWER
Contamination, non-specific binding, stop codon in primers, mastermix [] error,
thermocycle program wrong
, How do we know if PCR worked/failed? - ANSWER 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? - ANSWER Checks the
purity of the sample (DNA)
What values are being measured during DNA spectrophotometry and what does
each value mean? - ANSWER - 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? - ANSWER 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? - ANSWER
DNA is negatively charged at neutral pH, therefore it moves from cathode (-) to
anode (+)
What is the purpose of agarose gel electrophoresis? - ANSWER Standard
method used to seperate, identify and purify DNA, RNA, and proteins
What components are needed for successful gel? What does each component
do? - ANSWER -TBE buffer in which the Tris component is a basic salt and
provides conductivity. Boric acid lowers pH of buffer, so DNA does not
hydrolyze, maintain correct charge to migrate through gel.
-EDTA is a chelating agent, binds to divalent cations like Mg2+ which is often
required by enzymes to degrade DNA
-Loading buffer to visualize the bands (blue/purple).
-DNA ladder to determine size (bp) of fragments in gel.
