Term
| What are the 5 molecular biology tools that we can use to image DNA? |
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Definition
| molecular separations, labels, blotting, sequencing and assaying protein-DNA interactions |
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Term
| What are 3 common molecular separation techniques? |
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Definition
1.Chromatography
2.Electrophoresis
3. Ultracentrifugation |
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Term
| What is ultracentrifugation? |
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Definition
| Centrifuging a sample at 150,000X Gravity |
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Term
| What is a specific type of UCF? |
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Definition
| Sucrose Gradient UCF and Equilibrium Density UCF |
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Term
| In Sucrose Gradient UCF, where is the most dense portion going to be? |
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Definition
| Dense-->near the bottom, lighter fragment near the top |
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Term
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Definition
Fragments will eventually stop moving because of equilibrium.
Smaller pieces will find an equilib near the top, larger near the bottom. |
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Term
| How does data come in in UCF? |
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Definition
| In a graph with peaks representing where and what fraction number is most concentrated (top or bottom) |
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Term
| What are 3 types of Gel electrophoresis? |
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Definition
1. DNA Gel electrophoreis (agarose/acrylamide or pulsed-field)
2. Protein Gel electrophoresis
3. 2D Gel electrophoresis |
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Term
| With DNA gel Electrophoresis, why must one load standards? |
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Definition
| fragments of a known size help you have a reference for your fragments |
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Term
| In DNA gel electrophoresis how does the data look? |
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Definition
Graph--X=distance migrated Y=fragment size
You want to measure the size of dna fragments |
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Term
| What is Pulsed-Field gel electrophoresis? |
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Definition
| Good for separating/resolving large fragments of DNA and raises resolution. Essentially the DNA fragments must change directions using an electric field |
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Term
| What is pulsed-field Gel electrophoresis good for? |
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Definition
| dealing with chromosome fragments of whole chromosomes |
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Term
| What does charge of a protein depend on? |
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Definition
| overall charge depends on Amino Acid composition and its shape. |
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Term
| In Protein Gel Electrophoresis what kind of gel is used? |
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Definition
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Term
| Why does it often contain SDS or Sodium dodecyl sulfate? |
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Definition
| this is a detergent to proteins and denatures them coating them with a uniform charge eliminating the charge and shape factor. Now the only variable left is the size. |
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Term
| What happens when you don't use SDS? |
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Definition
| you must consider charge and shape. If the protein is negatively charged, it wont go to negative pole, instead positive pole |
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Term
| What are 3 types of Chromatography? |
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Definition
1.Ion-exchange chromatography
2. Gel filtration chromatography
3. Affinity chromatography |
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Term
| What is the basis of Ion-Exchange chromatography ? |
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Definition
| Separate substances (usually proteins) according to charge via a resin |
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Term
| What is the first step in Ion-Exchange chromatography ? |
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Definition
Determine the charge of the protein using 1 positive and 1 negative column and figure out where the protein sticks.
This is called ANION/CATION Exchange |
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Term
| What are the steps for Ion-Exchange chromatography ? |
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Definition
1. Determine charge of protein using differently charged tubes.
2. Anion(neg) or cation(pos) exchagne
3. Add a salt (strong ions) in a gradient to compete with your protein (elute with salt gradient) |
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Term
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Definition
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Term
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Definition
| pH +Charge of protein A
pH>pI --> -Charge of protein A |
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Term
| What is Gel Filtration Chromatography? |
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Definition
| Separate substances based on physical dimensions (size) |
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Term
| What are the steps to gel filtration Chromatography? |
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Definition
| Add porous resins and determine what relative concentrations there are at different fraction numbers... |
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Term
| What passes through faster in Gel filtration chromatography? Small or Large? |
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Definition
| Large molecules move faster b/c they are excluded and emerge at void volume |
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Term
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Definition
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Term
| In Gel filtration Chromatography, what molecules move slower? |
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Definition
| Smaller molecules b/c they have access to space larger ones dont |
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Term
| Affinity Chromatography is a similar method as in creating what? |
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Definition
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Term
| What types of labels are available? |
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Definition
| Radioactive and Non-radioactive. |
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Term
| What are advantages/disadvantages to using radioactive labels? |
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Definition
Advantage: Can detect in small quantities
Disadvantage: health risk |
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Term
| What are radioactive labels used in? |
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Definition
| Autoradiography and liquid scintillation counting |
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Term
| What are advantages/disadvantages to using non-radioactive labels? |
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Definition
Advantage: Non-radioactive
Disadvantage: Less signal (must be enhanced) |
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Term
| What are some examples of Non-radioactive labels? |
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Definition
| Chromogenic (colorgenic) or Chemilluminescent (light-giving off) |
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Term
| In Autoradiography/phosphorimaging what are some commonly used isotopes? |
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Definition
35S, 32P (short half life--weeks/days)
125I (long half life)
3H(tritium) or 14C |
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Term
| How does Autoradiography/phosphorimaging work? |
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Definition
1.Electrophoresis with radioactive tags, gel becomes radioactive.
2. Expose to film on the gel for a period of time
3. develop film |
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Term
| What are 2 types of Nucleic Acid Hybridizations? |
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Definition
| Southern and Northern Blots |
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Term
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Definition
| Method to identify specific DNA fragments |
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Term
| In S. blots how are specific bands identified? |
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Definition
| labeled probes that are based on hybridzation/complentarity b/w DNA strands |
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Term
| In S. blots what must the Target DNA be in order for the DNA to bind on the probe? |
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Definition
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Term
| What is the method of S. Blot? |
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Definition
1. Separate by size by running a gel
2. Transfer to nylon membrane
a. Either by diffusion or by an electric current
3. End up with a piece of filter paper (blot) that contains the DNA fragments
4. May incubate in a buffer
5. Then incubate labled probe which is specific for sequence you are looking for
6. Then you will see where the probe has detected the blot
Probe=short DNA sequence that has radioactive tags |
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Term
| What is the shorthand version of S. Blots? |
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Definition
1. Separate
2.Transfer to membrane (diffusion or current)
3.Filter paper has DNA fragments
4. Incubate in buffer
5. Incubate labeled probe
6. Then you can see where probe detected blot
STFIIT |
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Term
| What is a probe in s. blot? |
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Definition
| Short DNA sequence that has radioactive tags. |
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Term
| What is important to note in S. Blot? |
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Definition
How stringent are you in getting every nucleotide to match? High stringency=needs near perfect match
Low Stringency=not 100% perfect match |
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Term
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Definition
| similar to S. blots but you are looking for RNA, most likely mRNA |
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Term
| How can you use N. Blots? |
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Definition
Looking for a gene involved in glycolysis in different tissues
mRNA or Gene expression levels
Lot of glycolysis in the tissues that are dark (heart and skeletal muscle is most)
Compare all sorts of tissue |
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Term
| What are Immunoblots (AKA W. Blots)? |
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Definition
| Detecting specific protein using an antibody |
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Term
| What are the steps in W. Blot? |
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Definition
1. Electrophoresis of proteins
a. Use SDS PAGE Gel (SDS polyacrylamide Gel)
2. Blot the proteins from the gel to a membrane (Nitrocellulose membrane to which proteins bind
3. Detect the protein using antibody or antiserum to the target protein
a. Antibodies are proteins that recognize and bind to a target protein
b. You can make an antibody yourself or outsource it…
i. The company will inject your proteins into an animal and then they will collect the blood and collect the antibodies
4. Labeled secondary antibody is used to bind the first antibody and increase the signal
a. Antibody made by us or by goats etc. there is a particular sequence that you can make antibodies to…
i. You can get an antibody that is anti goat or anti-mouse
ii. This is sort of specific binding it will bind to all antibodies by that organism
b. This will be the signal you will detect. |
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Term
| What is the shorthand of a W. Blot? |
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Definition
1. Electrophoresis
2.Blot proteins from Gel to membrane
3. Detect using Antibody
4. Labeled Second antibody to bind to 1st one
5. Detect Antibody
W.EBDLD |
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Term
| What is a method of DNA sequencing? |
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Definition
| Sanger Chain Termination Method |
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Term
| What do you need for the Sanger Chain Termination Method? |
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Definition
1. DNA template to be sequenced
2. Complementary Oligo (primer)
3. DNA Polymerase (Klenow Fragment)
a. Free 3'hydroxyl
4. Radioactive label
5. Exces sof normal deoxy nucleotides (dNTP)
6. Dideoxy nucleotides (ddATP, ddCTP, ddGTP, ddTTP)
a. Aka chain terminator
b. Terminates chain production b/c there is nothing to bind to--NO 3' Hydroxyl so polymerase cannot add any more nucleotides |
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Term
| Shorthand of Sanger Chain Termination Method |
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Definition
1.DNA Template
2.Complementary Oligo
3.DNA Polymerase
4.Radioactive Label
5.Excess of normal DNA
6. Chain Terminators
CTM: DCDREC |
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Term
| What are some tricks to the Sanger Chain Termination Method? |
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Definition
| Perform DNA syntehsis reactions with each chain termination in 4 different tubes |
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Term
| In Chain Term Method Gel, where is the 5' end? |
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Definition
| 5'-->3' = bottom-->top = template sequence |
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Term
| In the "old fashioned" how many nucleotides are you limited to read in a sequence? |
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Definition
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Term
| How does Automated DNA sequencing help? |
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Definition
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Term
| How many nucleotides are you limited to for the Automated Chain termination method? |
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Definition
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Term
| How is the automated method set up? |
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Definition
| Same thing as before, just you have a laser that emits a fluorescent light that is detected by a detector and then analyzed by a computer to give you nice sharp peaks |
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Term
| What are 3 ways to determine protein-DNA interactions? |
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Definition
1.Nitrocellulose filter binding
2. Gel mobility shift assay (EMSA)
3. Footprinting |
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Term
| What is important in footprinting? |
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Definition
1.DNAse
2.DMS-DNA methylating agent
3.Hydroxyl Radical |
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Term
| In Nitrocellulose filter binding Assay, what molecule do you label? protein or DNA? |
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Definition
| Label the DNA b/c the protein will always bind, the only way the DNA will bind to the Nitro paper is if it is bound to a protein |
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Term
| Describe steps in Nitrocellulose Filter Binding Assay. |
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Definition
1. Nitrocellulose binds protein, but not double stranded DNA and DNA will end up in the filtrate
2. Protein binds extremely well (there is nothing in the tube b/c the protein bound)
3. Protein-DNA complex DNA remains on the filter b/c the protein is bound to it
FREE DNA will not bind, it will remain in the filtrate |
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Term
| Describe steps in Nitrocellulose Filter Binding Assay. |
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Definition
Set up 3 tubes
1. Nitrocellulose binds protein, but not double stranded DNA and DNA will end up in the filtrate
2. Protein binds extremely well (there is nothing in the tube b/c the protein bound)
3. Protein-DNA complex DNA remains on the filter b/c the protein is bound to it
FREE DNA will not bind, it will remain in the filtrate |
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Term
| What is the Gel Mobility Shift Assay? |
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Definition
| As protein conc increases, there is less and less bare DNA to move through the gel |
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Term
| What is DNAse footprinting? |
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Definition
| DNAse not destroying a portion of DNA with protein bound to it so you can see the exact location of the protein, not just if there is protein or not. |
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Term
| Steps in DNAse footprinting: |
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Definition
1.Attain strand of DNA
2.Bind Protein
3.Add DNAse (low conc) then remove protein and denature DNA
4.Use electrophoresis to see the gel
5. locate sequence where protein bound
DNA-BAEL |
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