BIMM 100 FINAL questions well
answered already passed
What is the purpose of Sanger Sequencing? - correct answer ✔✔ To find the order of
nucleotides in an RNA/DNA sequence
How is sequencing done? - correct answer ✔✔ -ddNTPs at low levels are incorporated into the
expanding DNA strand
-when a ddNTP is added, DNA pol no longer has that 3'OH end to add to another nucleotide, so
the strand is terminated
-If the ddNTPs fluoresce we can connect the color that each strand glows with the ddNTP that
ended that strand
-the 5' end is at the bottom of the gel because it would be found at the lightest end of the
strand
What is the purpose of Gel Electrophoresis? - correct answer ✔✔ Separates strands of
nucleotides (DNA/RNA) by size
What is the purpose of Electrophoretic Mobile Shift Assay (EMSA)? - correct answer ✔✔ -To
find out if there is protein attached to a DNA strand
-the more protein you have with DNA, the heavier the sample is, and the slower it will migrate
to the bottom of the gel (the higher the band ends up, the more protein was in the sample)
What is the purpose of DNAse footprinting? - correct answer ✔✔ -To see where the
proteins/TF's are bound on a DNA strand
How is DNAse footprinting done? - correct answer ✔✔ -Using the enzymes DNAse, you can
chop up every single 'naked' or 'exposed' piece of DNA
,-the segment of DNA with the TF/protein bound will show up as a gap on an agarose gel; this is
what the 'foot print' is
How can you use antibodies to label proteins? - correct answer ✔✔ -binds to a specific part of a
protein
-can attach fluorescent chemicals to the ends of the antibodies to make any protein fluoresce
-antibodies are a type of label that are used to label proteins when conducting a Western Blot
How are probes used as labels? - correct answer ✔✔ -they label proteins of interest
-analogous to antibodies, BUT are made out of DNA or RNA and instead of using fluorescence,
probes usually use radiation
-probes will compliment pair (or hybridize) with the RNA sequence of interest
What is the purpose of a Western Blot? - correct answer ✔✔ -To detect a specific protein of
interest from a collection of proteins
-Western blot uses antibodies to label proteins
What antibody is commonly used as an example for a control group? - correct answer ✔✔ -IgG
= ab that is normally produced by our secondary immune response in response to a pathogen
-In vitro ---> it is often used against your input proteins as a CONTROL because IgG will bind to
NOTHING (should show no band on gel)
What is Chromatin Immunoprecipitation (ChIP)? - correct answer ✔✔ -Used to see where and
how much a protein of interest is bound to a specific sequence of DNA
How is a ChIP experiment done? - correct answer ✔✔ 1. Cells are fixed with formaldehyde to
cross-link histone and non-histone proteins to DNA
2. Chromatin is digested with micrococcal nuclease into 150-900 bp DNA/protein fragments
, 3. AB specific to histone of non-histone proteins are added and the complex co-precipitates and
is captured by protein G agarose or protein G magnetic beads
4. Cross-links are reversed and DNA is purified and ready for analysis
What is the signal that recruits RNA Pol II and defines where the transcription of a gene can
begin? - correct answer ✔✔ The promoter
What would happen if you add an inhibitor that targets TBP and makes it bind less well to the
promoter? - correct answer ✔✔ There would be an overall decrease in transcription (RNA
prod.)
What experiment would you use to measure a decrease in RNA product? - correct answer ✔✔
Northern Blot; use a probe specific to that DNA molecule
Reporter Gene Assay - correct answer ✔✔ -A plasmid is transformed with the gene of interest,
followed with the incorporation of a reporter gene (ie: GFP, luciferase, etc.) and then ran on an
agarose gel to observe gene expression levels; intensity of reporter gene will translate into
expression levels.
-can be used to determine if a specific protein functions as a repressor
What are two examples of controls used in a ChIP experiment? - correct answer ✔✔ 1.
Incorporate a non-specific ab (ie: IgG), which is not expected to associate with chromatin
specifically
2. Control the genomic region where you would not find your protein of interest
hat >ie: TFIID, which binds to the TATA box, would not be expected to bind outside of the
promoter region
answered already passed
What is the purpose of Sanger Sequencing? - correct answer ✔✔ To find the order of
nucleotides in an RNA/DNA sequence
How is sequencing done? - correct answer ✔✔ -ddNTPs at low levels are incorporated into the
expanding DNA strand
-when a ddNTP is added, DNA pol no longer has that 3'OH end to add to another nucleotide, so
the strand is terminated
-If the ddNTPs fluoresce we can connect the color that each strand glows with the ddNTP that
ended that strand
-the 5' end is at the bottom of the gel because it would be found at the lightest end of the
strand
What is the purpose of Gel Electrophoresis? - correct answer ✔✔ Separates strands of
nucleotides (DNA/RNA) by size
What is the purpose of Electrophoretic Mobile Shift Assay (EMSA)? - correct answer ✔✔ -To
find out if there is protein attached to a DNA strand
-the more protein you have with DNA, the heavier the sample is, and the slower it will migrate
to the bottom of the gel (the higher the band ends up, the more protein was in the sample)
What is the purpose of DNAse footprinting? - correct answer ✔✔ -To see where the
proteins/TF's are bound on a DNA strand
How is DNAse footprinting done? - correct answer ✔✔ -Using the enzymes DNAse, you can
chop up every single 'naked' or 'exposed' piece of DNA
,-the segment of DNA with the TF/protein bound will show up as a gap on an agarose gel; this is
what the 'foot print' is
How can you use antibodies to label proteins? - correct answer ✔✔ -binds to a specific part of a
protein
-can attach fluorescent chemicals to the ends of the antibodies to make any protein fluoresce
-antibodies are a type of label that are used to label proteins when conducting a Western Blot
How are probes used as labels? - correct answer ✔✔ -they label proteins of interest
-analogous to antibodies, BUT are made out of DNA or RNA and instead of using fluorescence,
probes usually use radiation
-probes will compliment pair (or hybridize) with the RNA sequence of interest
What is the purpose of a Western Blot? - correct answer ✔✔ -To detect a specific protein of
interest from a collection of proteins
-Western blot uses antibodies to label proteins
What antibody is commonly used as an example for a control group? - correct answer ✔✔ -IgG
= ab that is normally produced by our secondary immune response in response to a pathogen
-In vitro ---> it is often used against your input proteins as a CONTROL because IgG will bind to
NOTHING (should show no band on gel)
What is Chromatin Immunoprecipitation (ChIP)? - correct answer ✔✔ -Used to see where and
how much a protein of interest is bound to a specific sequence of DNA
How is a ChIP experiment done? - correct answer ✔✔ 1. Cells are fixed with formaldehyde to
cross-link histone and non-histone proteins to DNA
2. Chromatin is digested with micrococcal nuclease into 150-900 bp DNA/protein fragments
, 3. AB specific to histone of non-histone proteins are added and the complex co-precipitates and
is captured by protein G agarose or protein G magnetic beads
4. Cross-links are reversed and DNA is purified and ready for analysis
What is the signal that recruits RNA Pol II and defines where the transcription of a gene can
begin? - correct answer ✔✔ The promoter
What would happen if you add an inhibitor that targets TBP and makes it bind less well to the
promoter? - correct answer ✔✔ There would be an overall decrease in transcription (RNA
prod.)
What experiment would you use to measure a decrease in RNA product? - correct answer ✔✔
Northern Blot; use a probe specific to that DNA molecule
Reporter Gene Assay - correct answer ✔✔ -A plasmid is transformed with the gene of interest,
followed with the incorporation of a reporter gene (ie: GFP, luciferase, etc.) and then ran on an
agarose gel to observe gene expression levels; intensity of reporter gene will translate into
expression levels.
-can be used to determine if a specific protein functions as a repressor
What are two examples of controls used in a ChIP experiment? - correct answer ✔✔ 1.
Incorporate a non-specific ab (ie: IgG), which is not expected to associate with chromatin
specifically
2. Control the genomic region where you would not find your protein of interest
hat >ie: TFIID, which binds to the TATA box, would not be expected to bind outside of the
promoter region
