BCH 5413- EXAM 1 QUESTIONS WITH
COMPLETE SOLUTIONS (LATEST
2024|2025 UPDATES)
Western Blot Controls - Answer-1. Housekeeping protein that doesn't change with
treatment
2. Quality of Sample
3. Other possible interactions with antibody
4. positive and negative experimental controls
Positive control - Answer-makes sure reagents worked correctly
Northern: probe for something you know will show a change
Western: load a lane withe recombinant protein you know will give signal
Negative control - Answer-tests to see if NO signal when required reagent is missing
Western: using secondary antibody only
Equal loading controls - Answer-did you really put the same amount of sample into each
lane?
probe for GAPDH or actin
Cross-reactions - Answer-Could one of your reagents be detecting something else as
well?
use BLAST (NCIB website)
Thermes Aquatic - Answer-heat stable polymerase (Taq polymerase)
PCR Denaturing - Answer-At 95 degrees C for 30 seconds
PCR annealing primers - Answer-between 30-50 degree C for 30 seconds
PCR extending primers - Answer-at 72 degree C for 1 minute
PCR reaction components - Answer-1. Template DNA
2. Primers (forward and reverse)
3. dNTP (G, A, C, T)
4. Taq polymerase
5. Buffer + magnesium
6. Water
Uses of PCR - Answer-1. Cloning
2. Genotyping (Triplex PCR)
, 3. Analyze DNA methylation patterns (Bisulfite PCR)
4. Characterize DNA and protein interactions (footprinting)
5. amplify rare footprint (Ligation-mediated PCR)
DNA Footprinting - Answer-used to analyze sequence-specific DNA-protein interactions
•DNA footprinting occurs when digestion of a specific region of DNA is prevented by
binding of a protein. The protection can be either to digestion by DNases, or to chemical
modification. The second process gives greater resolution.
***The footprint represents the region of
DNA protected by the protein, and therefore tells where
the protein binds
Ligation-mediated PCR - Answer-used to AMPLIFY rare footprinted fragment by a PCR
step followed by blotting and probing
Taqman - Answer-2 primers, 1 probe
allows amplification of two sequences but is expensive and labor intensive
SYBR - Answer-dsDNA binding dye
monitors amplification of any ds DNA (no probe required), cheap, easy to design but
nonspeicific and may generate false positive
CT: - Answer-when the cycle number crosses a threshold, lower CT means more
product is present
Restriction endonucleases - Answer-Cut dsDNA into inverted palindromes
HindII sequence recognition - Answer-GTPyPuAC
CAPuPyTG
HindIII sequence recognition - Answer-A ↓ A G C T T
EcoR1 recognition sequence - Answer-G ↓ A A T T C
BamH1 recognition sequence - Answer-G ↓ G A T C C
hemimethylated state of DNA - Answer-protected against cleavage by restriction
endonuclease
first artificial plasmid - Answer-pBR322
Components of Plasmid - Answer-1. OriC (origin of replication)
2. Selectable marker either Ap ^R (ampicillin resistance) or Tc ^R (tetracycline
resistance)
COMPLETE SOLUTIONS (LATEST
2024|2025 UPDATES)
Western Blot Controls - Answer-1. Housekeeping protein that doesn't change with
treatment
2. Quality of Sample
3. Other possible interactions with antibody
4. positive and negative experimental controls
Positive control - Answer-makes sure reagents worked correctly
Northern: probe for something you know will show a change
Western: load a lane withe recombinant protein you know will give signal
Negative control - Answer-tests to see if NO signal when required reagent is missing
Western: using secondary antibody only
Equal loading controls - Answer-did you really put the same amount of sample into each
lane?
probe for GAPDH or actin
Cross-reactions - Answer-Could one of your reagents be detecting something else as
well?
use BLAST (NCIB website)
Thermes Aquatic - Answer-heat stable polymerase (Taq polymerase)
PCR Denaturing - Answer-At 95 degrees C for 30 seconds
PCR annealing primers - Answer-between 30-50 degree C for 30 seconds
PCR extending primers - Answer-at 72 degree C for 1 minute
PCR reaction components - Answer-1. Template DNA
2. Primers (forward and reverse)
3. dNTP (G, A, C, T)
4. Taq polymerase
5. Buffer + magnesium
6. Water
Uses of PCR - Answer-1. Cloning
2. Genotyping (Triplex PCR)
, 3. Analyze DNA methylation patterns (Bisulfite PCR)
4. Characterize DNA and protein interactions (footprinting)
5. amplify rare footprint (Ligation-mediated PCR)
DNA Footprinting - Answer-used to analyze sequence-specific DNA-protein interactions
•DNA footprinting occurs when digestion of a specific region of DNA is prevented by
binding of a protein. The protection can be either to digestion by DNases, or to chemical
modification. The second process gives greater resolution.
***The footprint represents the region of
DNA protected by the protein, and therefore tells where
the protein binds
Ligation-mediated PCR - Answer-used to AMPLIFY rare footprinted fragment by a PCR
step followed by blotting and probing
Taqman - Answer-2 primers, 1 probe
allows amplification of two sequences but is expensive and labor intensive
SYBR - Answer-dsDNA binding dye
monitors amplification of any ds DNA (no probe required), cheap, easy to design but
nonspeicific and may generate false positive
CT: - Answer-when the cycle number crosses a threshold, lower CT means more
product is present
Restriction endonucleases - Answer-Cut dsDNA into inverted palindromes
HindII sequence recognition - Answer-GTPyPuAC
CAPuPyTG
HindIII sequence recognition - Answer-A ↓ A G C T T
EcoR1 recognition sequence - Answer-G ↓ A A T T C
BamH1 recognition sequence - Answer-G ↓ G A T C C
hemimethylated state of DNA - Answer-protected against cleavage by restriction
endonuclease
first artificial plasmid - Answer-pBR322
Components of Plasmid - Answer-1. OriC (origin of replication)
2. Selectable marker either Ap ^R (ampicillin resistance) or Tc ^R (tetracycline
resistance)
